Ribosome structure and protein synthesis inhibitors

ABSTRACT

The invention provides methods for producing high resolution crystals of ribosomes and ribosomal subunits as well as crystals produced by such methods. The invention also provides high resolution structures of ribosomal subunits either alone or in combination with protein synthesis inhibitors. The invention provides methods for identifying ribosome-related ligands and methods for designing ligands with specific ribosome-binding properties as well as ligands that may act as protein synthesis inhibitors. Thus, the methods and compositions of the invention may be used to produce ligands that are designed to specifically kill or inhibit the growth of any target organism.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.09/635,708, filed Aug. 9, 2000, and claims the benefit of (i) U.S.Provisional Application No. 60/223,977, filed Aug. 9, 2000, (ii) U.S.Provisional Application No. [Atty. Docket No. RIB-002PR], entitled “TheKink-Turn: a New RNA Secondary Structure Motif,” filed Jul. 20, 2001,and (iii) U.S. Provisional Application No. [Atty. Docket No.RIB-002PR2], entitled “The Kink-Turn: a New RNA,” filed Aug. 1, 2001,the disclosures of each of the foregoing of which are incorporated byreference herein.

GOVERNMENT LICENSE RIGHTS

Certain work described herein was supported, in part, by Federal GrantNos. NIH-GM22778 and NIH-GM54216, awarded by the National Institutes ofHealth. The Government may have certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to the field of proteinbiosynthesis and to modulators, for example, inhibitors, of proteinbiosynthesis. More particularly, the invention relates to methods andcompositions for elucidating the three-dimensional structure of thelarge ribosomal subunit, either alone or in combination with a proteinsynthesis inhibitor; the three-dimensional structure of the largeribosomal subunit, either alone or in combination with a proteinsynthesis inhibitor; the use of such structures in the design andtesting of novel protein synthesis inhibitors; and novel proteinsynthesis inhibitors.

BACKGROUND

I. Ribosomes: Structure, Function, and Composition

Ribosomes are ribonucleoproteins which are present in both prokaryotesand eukaryotes. They comprise about two-thirds RNA and one-thirdprotein. Ribosomes are the cellular organelles responsible for proteinsynthesis. During gene expression, ribosomes translate the geneticinformation encoded in a messenger RNA into protein (Garrett et al.(2000) “The Ribosome: Structure, Function, Antibiotics and CellularInteractions,” American Society for Microbiology, Washington, D.C.).

Ribosomes comprise two nonequivalent ribonucleoprotein subunits. Thelarger subunit (also known as the “large ribosomal subunit”) is abouttwice the size of the smaller subunit (also known as the “smallribosomal subunit”). The small ribosomal subunit binds messenger RNA(mRNA) and mediates the interactions between mRNA and transfer RNA(tRNA) anticodons on which the fidelity of translation depends. Thelarge ribosomal subunit catalyzes peptide bond formation—thepeptidyl-transferase reaction of protein synthesis—and includes (atleast) two different tRNA binding sites: the A-site which accommodatesthe incoming aminoacyl-tRNA, which is to contribute its amino acid tothe growing peptide chain, and the P-site which accommodates thepeptidyl-tRNA complex, i.e., the tRNA linked to all the amino acids thathave so far been added to the peptide chain. The large ribosomal subunitalso includes one or more binding sites for G-protein factors thatassist in the initiation, elongation, and termination phases of proteinsynthesis. The large and small ribosomal subunits behave independentlyduring the initiation phase of protein synthesis; however, they assembleinto complete ribosomes when elongation is about to begin.

The molecular weight of the prokaryotic ribosome is about 2.6×10⁶daltons. In prokaryotes, the small ribosomal subunit contains a 16S(Svedberg units) ribosomal RNA (rRNA) having a molecular weight of about5.0×10⁵ daltons. The large ribosomal subunit contains a 23S rRNA havinga molecular weight of about 1.0×10⁶ daltons and a 5S rRNA having amolecular weight of about 4.0×10⁵ daltons. The prokaryotic small subunitcontains about 20 different proteins and its large subunit containsabout 35 proteins. The large and small ribosomal subunits togetherconstitute a 70S ribosome in prokaryotes.

Eukaryotic ribosomes generally are bigger than their prokaryoticcounterparts. In eukaryotes, the large and small subunits together makean 80S ribosome. The small subunit of a eukaryotic ribosome includes asingle 18S rRNA, while the large subunit includes a 5S rRNA, a 5.8SrRNA, and a 28S rRNA. The 5.8S rRNA is structurally related to the 5′end of the prokaryotic 23S rRNA, and the 28S rRNA is structurallyrelated to the remainder of the prokaryotic 23S rRNA (Moore (1998) Annu.Rev. Biophys. 27: 35-58). Eukaryotic ribosomal proteins arequalitatively similar to the prokaryotic ribosomal proteins; however,the eukaryotic proteins are bigger and more numerous (Moore (1998)supra).

II. Structural Conservation of the Large Ribosomal Subunit

While the chemical composition of large ribosomal subunits vary fromspecies to species, the sequences of their components provideunambiguous evidence that they are similar in three-dimensionalstructure, function in a similar manner, and are related evolutionarily.The evolutionary implications of rRNA sequence data available arereviewed in the articles of Woese and others in part II of RibosomalRNA. Structure, Evolution, Processing and Function in ProteinBiosynthesis, (Zimmermann and Dahlberg, eds., CRC Press, Boca Raton,Fla., 1996). The article by Garret and Rodriguez-Fonseca in part IV ofthe same volume discusses the unusually high level of sequenceconservation observed in the peptidyl transferase region of the largeribosomal subunit. The ribosomes of archeal species like Haloarculamarismortui resemble those obtained from eubacterial species like E.coli in size and complexity. However, the proteins in H. marismortuiribosomes are more closely related to the ribosomal proteins found ineukaryotes (Wool et al. (1995) Biochem. Cell Biol. 73: 933-947).

III. Determination of the Structure of Ribosomes

Much of what is known about ribosome structure is derived from physicaland chemical methods that produce relatively low-resolution information.Electron microscopy (EM) has contributed to an understanding of ribosomestructure ever since the ribosome was discovered. In the 1970s, lowresolution EM revealed the shape and quaternary organization of theribosome. By the end of 1980s, the positions of the surface epitopes ofall the proteins in the E. coli small subunit, as well as many in thelarge subunit, had been mapped using immunoelectron microscopytechniques (Oakes et al. (1986), Structure Function and Genetics ofRibosomes, (Hardesty, B. and Kramer, G., eds.) Springer-Verlag, NewYork, N.Y., pp. 47-67; Stoeffler et al. (1986), Structure, Function andGenetics of Ribosomes, (Hardesty, B. and Kramer, G., eds.)Springer-Verlag, New York, N.Y., pp. 28-46). In the last few years,advances in single-particle cryo-EM and image reconstruction have led tothree-dimensional reconstructions of the E. coli 70S ribosome and itscomplexes with tRNAs and elongation factors to resolutions of between 15Å and 25 Å (Stark et al. (1995) Structure 3: 815-821; Stark et al.(1997) Nature 3898: 403-406; Agrawal et al. (1996) Science 271:1000-1002; Stark et al. (1997) Cell 28: 19-28). Additionally,three-dimensional EM images of the ribosome have been produced atresolutions sufficiently high so that many of the proteins and nucleicacids that assist in protein synthesis can be visualized bound to theribosome. An approximate model of the RNA structure in the large subunithas been constructed to fit a 7.5 Å resolution electron microscopic mapof the 50S subunit from E. coli and available biochemical data (Muelleret al. (2000) J. Mol. Biol. 298: 35-59).

While the insights provided by EM have been useful, it has long beenrecognized that a full understanding of ribosome structure would deriveonly from X-ray crystallography. In 1979, Yonath and Wittman obtainedthe first potentially useful crystals of ribosomes and ribosomalsubunits (Yonath et al. (1980) Biochem. Internal. 1: 428-435). By themid 1980s, scientists-were preparing ribosome crystals for X-raycrystallography (Maskowski et al. (1987) J. Mol. Biol. 193: 818-822).The first crystals of 50S ribosomal subunit from H. marismortui wereobtained in 1987. In 1991, improvements were reported in the resolutionof the diffraction data obtainable from the crystals of the 50Sribosomal subunit of H. marismortui (van Bohlen, K. (1991) J. Mol. Biol.222: 11).

In 1995, low resolution electron density maps for the large and smallribosomal subunits from halophilic and thermophilic sources werereported (Schlunzen et al. (1995) Biochem. Cell Biol. 73: 739-749).However, these low resolution electron density maps proved to bespurious (Ban et al. (1998) Cell 93: 1105-1115).

The first electron density map of the ribosome that showed featuresrecognizable as duplex RNA was a 9 Å resolution X-ray crystallographicmap of the large subunit from Haloarcula marismortui (Ban et al. (1998)supra). Extension of the phasing of that map to 5 Å resolution made itpossible to locate several proteins and nucleic acid sequences, thestructures of which had been determined independently (Ban et al. (1999)Nature 400: 841-847).

At about the same time, using similar crystallographic strategies, a 7.8Å resolution map was generated of the entire Thermus thermophilusribosome showing the positions of tRNA molecules bound to its A-, P-,and E-(protein exit site) sites (Cate et al. (1999) Science 285:2095-2104), and a 5.5 Å resolution map of the 30S subunit from Tthermophilus was obtained that allowed the fitting of solved proteinstructures and the interpretation of some of its RNA features (Clemons,Jr. et al. (1999) Nature 400: 833-840). Subsequently, a 4.5 Å resolutionmap of the T. thermophilus 30S subunit was published, which was based inpart on phases calculated from a model corresponding to 28% of thesubunit mass that had been obtained using a 6 Å resolution experimentalmap (Tocilj et al. (1999) Proc. Natl. Acad. Sci. USA 96: 14252-14257).

IV. Location of the Peptidyl Transferase Site in the Large RibosomalSubunit

It has been known for about 35 years that the peptidyl transferaseactivity responsible for the peptide bond formation that occurs duringmessenger RNA-directed protein synthesis is intrinsic to the largeribosomal subunit (Traut et al. (1964) J. Mol. Biol. 10: 63; Rychlik(1966) Biochini. Biophys. Acta 114: 425; Monro (1967) J. Mol. Biol. 26:147-151; Maden et al. (1968) J. Mol. Biol. 35: 333-345) and it has beenunderstood for even longer that the ribosome contains proteins as wellas RNA. In certain species of bacteria, for example, the large ribosomalsubunit contains about 35 different proteins and two RNAs (Noller (1984)Ann. Rev. Biochem. 53: 119-162; Wittmann-Liebold et al. (1990) TheRibosome: Structure Function, and Evolution, (W. E. Hill et al., eds.)American Society for Microbiology, Washington, D.C. (1990), pp.598-616). These findings posed three related questions. Which of thealmost 40 macromolecular components of the large ribosomal subunitcontribute to its peptidyl transferase site, where is that site locatedin the large subunit, and how does it work?

By 1980, the list of components that might be part of the ribosome'speptidyl transferase had been reduced to about half a dozen proteins and23S rRNA (see Cooperman (1980) Ribosomes: Structure, Function andGenetics, (G. Chambliss et al., eds.) University Park Press, Baltimore,Md. (1980), 531-554), and following the discovery of catalytic RNAs(Guerrier-Takada et al. (1983) Cell 35: 849-857; Kruger et al. (1982)Cell 31: 147-157), the hypothesis that 23S rRNA might be its soleconstituent, which had been proposed years earlier, began to gain favor.In 1984, Noller and colleagues published affinity labeling results whichshowed that U2619 and U2620 (in E. coli: U2584, U2585) are adjacent tothe CCA-end of P-site-bound tRNA (Barta et al. (1984) Proc. Nat. Acad.Sci. USA 81: 3607-3611; Vester et al. (1988) EMBO J. 7: 3577-3587).These nucleotides appear to be part of a highly conserved internal loopin the center of domain V of 23S rRNA. The hypothesis that this loop isintimately involved in the peptidyl transferase activity was supportedby the observation that mutations in that loop render cells resistant tomany inhibitors of peptidyl transferase, and evidence implicating it inthis activity has continued to mount (see, Noller (1991) Ann. Rev.Biochem. 60: 191-227; Garrett et al. (1996) Ribosomal RNA: Structure,Evolution, Processing and Function in Protein Biosynthesis, (R. A.Zimmerman and A. E. Dahlberg, eds.) CRC Press, Boca Raton, Fla. (1996),pp. 327-355).

Definitive proof that the central loop in domain V is the sole componentof the ribosome involved in the peptidyl transferase activity hasremained elusive, however. Studies have shown that it was possible toprepare particles that retained peptidyl transferase activity byincreasingly vigorous deproteinizations of large ribosomal subunits,however, it was not possible to produce active particles that werecompletely protein-free. Nevertheless, combined with earlierreconstitution results (Franceschi et al. (1990) J. Biol. Chem. 265:6676-6682), this work reduced the number of proteins that might beinvolved to just two: L2 and L3 (see, Green et al. (1997) Annu. Rev.Biochem. 66: 679-716). More recently, Watanabe and coworkers reportedsuccess in eliciting peptidyl transferase activity from in vitrosynthesized, protein-free 23S rRNA (Nitta et al. (1998) RNA 4: 257-267),however, their observations appear not to have withstood furtherscrutiny. Thus the question still remained: is the ribosome a ribozymeor is it not?

Over the years, the location of the peptidyl transferase site in theribosome has been approached almost exclusively by electron microscopy.In the mid-1980s evidence that there is a tunnel running through thelarge ribosomal subunit from the middle of its subunit interface side toits back (Milligan et al. (1986) Nature 319: 693-695; Yonath et al.(1987) Science 236: 813-816) began to accumulate, and there has beenstrong reason to believe that polypeptides pass through it as they aresynthesized (Bemabeu et al. (1982) Proc. Nat. Acad. Sci. USA 79:3111-3115; Ryabova et al. (1988) FEBS Letters 226: 255-260; Beckmann etal. (1997) Science 278: 2123-2126). More recent cryo-EM investigations(Frank et al. (1995) Nature 376: 441-444; Frank et al. (1995) Biochemn.Cell Biol. 73: 757-765; Stark et al. (1995) supra) confirmed theexistence of the tunnel and demonstrated that the CCA-ends ofribosome-bound tRNAs bound to the A- and P-sites are found in thesubunit interface end of the tunnel. Consequently, the peptidyltransferase site must be located at that same position, which is at thebottom of a deep cleft in the center of the subunit interface surface ofthe large subunit, immediately below its central protuberance.

The substrates of the reaction catalyzed at the peptidyl transferasesite of the large subunit are an aminoacyl-tRNA (aa-tRNA) and apeptidyl-tRNA. The former binds in the ribosome's A-site and the latterin its P-site. The α-amino group of the aa-tRNA attacks the carbon ofthe carbonyl acylating the 3′ hydroxyl group of the peptidyl-tRNA, and atetrahedral intermediate is formed at the carbonyl carbon. Thetetrahedral intermediate resolves to yield a peptide extended by oneamino acid esterified to the A-site bound tRNA and a deacylated tRNA inthe P-site.

This reaction scheme is supported by the observations of Yarus andcolleagues who synthesized an analogue of the tetrahedral intermediateby joining an oligonucleotide having the sequence CCdA to puromycin viaa phosphoramide group (Welch et al. (1995) Biochemistry 34: 385-390).The sequence CCA, which is the 3′ terminal sequence of all tRNAs, bindsto the large subunit by itself, consistent with the biochemical datashowing that the interactions between tRNAs and the large subunitlargely depend on their CCA sequences (Moazed et al. (1991) Proc. Natl.Acad. Sci. USA 88: 3725-3728). Puromycin is an aa-tRNA analogue thatinteracts with the ribosomal A-site, and the phosphoramide group of thecompound mimics the tetrahedral carbon intermediate. This transitionstate analogue, CCdA-phosphate-puromycin (CCdA-p-Puro), binds tightly tothe ribosome, and inhibits its peptidyl transferase activity (Welch etal. (1995) supra).

V. Structure Determination of Macromolecules Using X-ray Crystallography

In order to better describe efforts undertaken to determine thestructure of ribosomes, a general overview of X-ray crystallography isprovided below.

Each atom in a crystal scatters X-rays in all directions, butcrystalline diffraction is observed only when a crystal is orientedrelative to the X-ray beam so that the atomic scattering interferesconstructively. The orientations that lead to diffraction may becomputed if the wavelength of the X-rays used and the symmetry anddimensions of the crystal's unit cell are known (Blundell et al. (1976)Protein Crystallography (Molecular Biology Series), Academic Press,London). The result is that if a detector is placed behind a crystalthat is being irradiated with monochromatic X-rays of an appropriatewavelength, the diffraction pattern recorded will consist of spots, eachspot representing one of the orientations that gives rise toconstructive interference.

Each spot in such a pattern, however it is recorded, is characterized by(i) an intensity (its blackness); (ii) a location, which encodes theinformation about diffraction orientation; and (iii) a phase. If all ofthose things are known about each spot in a crystal diffraction pattern,the distribution of electrons in the unit cell of the crystal may becomputed by Fourier transformation (Blundell et al. (1976) supra), andfrom that distribution or electron density map, atomic positions can bedetermined.

Unfortunately, the phase information essential for computing electrondistributions cannot be measured directly from diffraction patterns. Oneof the methods routinely used to determine the phases of macromolecules,such as proteins and nucleic acids, is called multiple isomorphousreplacement (MIR) which involves the introduction of new X-rayscatterers into the unit cell of the crystal. Typically, these additionsare heavy atoms, which make a significant contribution to thediffraction pattern. It is important that the additions be sufficientlylow in number so that their positions can be located and that they leavethe structure of the molecule or of the crystal cell unaltered, i.e.,the crystals should be isomorphous. Isomorphous replacement usually isperformed by diffusing different heavy-metal complexes into the channelsof the preformed protein crystals. Macromolecules expose side chains(such as SH groups) in these solvent channels that are able to bindheavy metals. It is also possible to replace endogenous light metals inmetalloproteins with heavier ones, e.g., zinc by mercury, or calcium bysamarium. Alternatively, the isomorphous derivative can be obtained bycovalently attaching a heavy metal to the macromolecule in solution andthen subjecting it to crystallization conditions.

Heavy metal atoms routinely used for isomorphous replacement include butare not limited to mercury, uranium, platinum, gold, lead, and selenium.Specific examples include mercury chloride, ethyl-mercury phosphate, andosmium pentamine, iridium pentamine. Since such heavy metals containmany more electrons than the light atoms (H, N, C, O, and S) of theprotein, the heavy metals scatter x-rays more strongly. All diffractedbeams would therefore increase in intensity after heavy-metalsubstitution if all interference were positive. In fact, however, someinterference is negative; consequently, following heavy-metalsubstitution, some spots increase in intensity, others decrease, andmany show no detectable difference.

Phase differences between diffracted spots can be determined fromintensity changes following heavy-metal substitution. First, theintensity differences are used to deduce the positions of the heavyatoms in the crystal unit cell. Fourier summations of these intensitydifferences give maps, of the vectors between the heavy atoms, theso-called Patterson maps. From these vector maps, the atomic arrangementof the heavy atoms is deduced. From the positions of the heavy metals inthe unit cell, the amplitudes and phases of their contribution to thediffracted beams of protein crystals containing heavy metals iscalculated.

This knowledge then is used to find the phase of the contribution fromthe protein in the absence of the heavy-metal atoms. As both the phaseand amplitude of the heavy metals and the amplitude of the protein aloneis known, as well as the amplitude of the protein plus heavy metals(i.e., protein heavy-metal complex), one phase and three amplitudes areknown. From this, the interference of the X-rays scattered by the heavymetals and protein can be calculated to determine if the interference isconstructive or destructive. The extent of positive or negativeinterference, with knowledge of the phase of the heavy metal, give anestimate of the phase of the protein. Because two different phase anglesare determined and are equally good solutions, a second heavy-metalcomplex can be used which also gives two possible phase angles. Only oneof these will have the same value as one of the two previous phaseangles; it therefore represents the correct phase angle. In practice,more than two different heavy-metal complexes are usually made in orderto give a reasonably good estimate of the phase for all reflections.Each individual phase estimate contains experimental errors arising fromerrors in the measured amplitudes. Furthermore, for many reflections,the intensity differences are too small to measure after one particularisomorphous replacement, and others can be tried.

The amplitudes and the phases of the diffraction data from the proteincrystals are used to calculate an electron-density map of the repeatingunit of the crystal. This map then is interpreted to accommodate theresidues of the molecule of interest. That interpretation is made morecomplex by several limitations in the data. First, the map itselfcontains errors, mainly due to errors in the phase angles. In addition,the quality of the map depends on the resolution of the diffractiondata, which, in turn, depends on how well-ordered the crystals are. Thisdirectly influences the quality of the map that can be produced. Theresolution is measured in angstrom units (A); the smaller this numberis, the higher the resolution and, therefore, the greater the amount ofdetail that can be seen.

Building the initial model is a trial-and-error process. First, one hasto decide how a polypeptide chain or nucleic acid weaves its way throughthe electron-density map. The resulting chain trace constitutes ahypothesis by which one tries to match the density of side chains to theknown sequence of the polypeptide or nucleic acid. When a reasonablechain trace has finally been obtained, an initial model is built thatfits the atoms of the molecule into the electron density. Computergraphics are used both for chain tracing and for model building topresent the data and manipulated the models.

The initial model will contain some errors. Provided the crystalsdiffract to high enough resolution (e.g., better than 3.5 Å), most orsubstantially all of the errors can be removed by crystallographicrefinement of the model using computer algorithms. In this process, themodel is changed to minimize the difference between the experimentallyobserved diffraction amplitudes and those calculated for a hypotheticalcrystal containing the model (instead of the real molecule). Thisdifference is expressed as an R factor (residual disagreement) which is0.0 for exact agreement and about 0.59 for total disagreement.

In general, the R factor for a well-determined macromolecular structurepreferably lies between 0.15 and 0.35 (such as less than about0.24-0.28). The residual difference is a consequence of errors andimperfections in the data. These derive from various sources, includingslight variations in the conformation of the protein molecules, as wellas inaccurate corrections both for the presence of solvent and fordifferences in the orientation of the microcrystals from which thecrystal is built. This means that the final model represents an averageof molecules that are slightly different both in conformation andorientation.

In refined structures at high resolution, there are usually no majorerrors in the orientation of individual residues, and the estimatederrors in atomic positions are usually around 0.1-0.2 Å, provided thesequence of the protein or nucleic acid is known. Hydrogen bonds, bothwithin the molecule of interest and to bound ligands, can be identifiedwith a high degree of confidence.

Typically, X-ray structures can be determined provided the resolution isbetter than 3.5 Å. Electron-density maps are interpreted by fitting theknown amino acid and/or nucleic acid sequences into regions of electrondensity.

VI. The Need for Higher Resolution for the 50S Ribosomal Subunit

Although the art provides crystals of the 50S ribosomal subunit, and 9 Åand 5 Å resolution X-ray crystallographic maps of the structure of the50S ribosome, the prior art crystals and X-ray diffraction data are notsufficient to establish the three-dimensional structures of all 31proteins and 3,043 nucleotides of the 50S ribosomal subunit. Thus, theprior art crystals and maps are inadequate for the structure-baseddesign of active agents, such as herbicides, drugs, insecticides, andanimal poisons.

More detailed, higher resolution X-ray crystallographic maps arenecessary in order to determine the location and three-dimensionalstructure of the proteins and nucleotides in ribosomes and ribosomalsubunits, particularly for the 50S ribosomal subunit. An accuratemolecular structure of the 50S ribosomal subunit will not only enablefurther investigation and understanding of the mechanism of proteinsynthesis, but also the development of effective therapeutic agents anddrugs that modulate (e.g., induce or inhibit) protein synthesis.

SUMMARY OF THE INVENTION

The present invention is based, in part, upon the determination of ahigh resolution atomic structure of a ribosomal subunit, moreparticularly, a large subunit of a ribosome. The high resolutionstructure has been determined for a large ribosomal subunit present inthe organism, Haloarcula marismortui. However, in view of the high levelof sequence and structural homology between ribosomes of organisms indifferent kingdoms, the structural information disclosed herein can beused to produce, using routine techniques, high resolution structuralmodels of large ribosomal units for any organism of interest.

Although there is significant homology between ribosomes of differentorganisms, for example, between ribosomes of humans and certain humanpathogens, there still are differences that can be exploitedtherapeutically. For example, many clinically and commerciallysignificant protein synthesis inhibitors, for example, antibiotics suchas streptomycin, tetracycline, chloramphenicol and erythromycin,selectively target bacterial ribosomes and disrupt bacterial proteinsynthesis but at the same time do not target or otherwise significantlyaffect human ribosome function. As a result, over the years antibioticshave proven to be invaluable in the treatment of microbial infections inhumans. However, there is still an ongoing need for new proteinsynthesis inhibitors, particularly because of the development of strainsof pathogens that are resistant to known antibiotics. The informationprovided herein provides insights into the design of new proteinsynthesis inhibitors.

The invention herein provides methods and compositions for resolving toa high resolution, the three-dimensional structure of a ribosomalsubunit of interest. In addition, the invention provides computersystems containing atomic co-ordinates that define at least a portion ofthe three-dimensional structure of a ribosome, more specifically, alarge ribosomal subunit. In addition, the invention provides methods ofusing the atomic co-ordinates to identify new molecules that canselectively bind ribosomes, and that preferably act as selectiveinhibitors of protein synthesis. In addition, the invention provides newfamilies of protein synthesis inhibitors. Each of these aspects of theinvention is discussed in more detail below.

In one aspect, the invention provides crystals, preferably untwinnedcrystals, of ribosomes or ribosomal subunits that have an averagethickness greater than about 15 μm. More specifically, the inventionprovides crystals having an average thickness from about 16 μm to about65 μm, or from about 66 μm to about 105 μm, or from about 104 μm toabout 155 μm, or from about 156 μm to about 205 μm. In particular, theinvention provides crystals having an average thickness from about 100μm to about 200 μm.

In a preferred embodiment, the invention provides crystals that have anaverage thickness greater than about 15 μm and/or are untwinned whereinthe crystals comprise the large ribosomal subunit. More particularly,the present invention provides such crystals wherein the large ribosomalsubunit is a 50S or 60S ribosomal subunit. The crystals may be obtainedusing the ribosomes or ribosomal subunits from prokaryotes or fromeukaryotes. In a preferred embodiment, the invention provides crystalscontaining ribosomes or ribosomal subunits obtained from bacteria orarchaebacteria, more particularly, from the organism Haloarculamarismortui. However, crystals may be obtained from ribosomes orribosomal subunits from any organism, particularly from animals, moreparticularly from mammals, and even more particularly from humans.

In another preferred embodiment, the crystals diffract X-rays to aresolution of at least about 4.5 Å, more preferably to a resolution ofat least about 3.0 Å, and most preferably to a resolution of about 2.4 Åfor the determination of atomic co-ordinates of ribosomes or ribosomalsubunits. In another preferred embodiment, the crystals of the presentinvention may also include a ligand, for example, a protein synthesisinhibitor, for example, an antibiotic, (such as a macrolide antibiotic)complexed with, or bound to a ribosome or ribosomal subunit.

In another aspect, the invention provides crystals of 50S ribosomalsubunits whose atomic structure is characterized by the atomicco-ordinates deposited at the Protein Data Bank ID: 1FFK or 1JJ2. Theinvention further provides phases computed from the co-ordinates of thedeposited co-ordinates and the uses of such phase information. In apreferred embodiment, the invention provides crystals of 50S ribosomalsubunits whose atomic structure is characterized by the atomicco-ordinates deposited at the Protein Data Bank ID: 1FFZ (largeribosomal subunit complexed with CCdA-p-Puro); or 1FG0 (large ribosomalsubunit complexed with a mini-helix analogue of aminoacyl-tRNA); as wellas those ribosomal subunits whose atomic structure is characterized bythe atomic coordinates listed in a file contained on Disk No. 3 of 3,specifically: large ribosomal subunit complexed with anisomycin (filename: anisomysin.pdb); large ribosomal subunit complexed withblasticidin (file name: blasticidin.pdb); large ribosomal subunitcomplexed with carbomycin (file name: carbomycin.pdb); large ribosomalsubunit complexed with tylosin (file name: tylosin.pdb); large ribosomalsubunit complexed with sparsomycin (file name: sparsomycin.pdb); largeribosomal subunit complexed with virginiamycin (file name:virginiamycin.pdb); or large ribosomal subunit complexed with spiramycin(file name: spiramycin.pdb).

In another embodiment, the invention provides a method of obtaining anelectron density map of a ribosomal subunit of interest that is onlyslightly different from the ribosomal subunit whose structure hasalready been determined, for example, by X-ray crystallography. Themethod comprises the steps of: (a) producing a crystal of the ribosomalsubunit of interest, wherein the crystal is isomorphous; (b) obtainingdiffraction amplitudes of the crystal produced in step (a); (c)combining the phases of the crystal of the ribosomal subunit whosestructure is already known with the diffraction amplitudes obtained instep (b) to produce a combined data set; and (d) obtaining an electrondensity map of the selected ribosomal subunit based on the combined dataset obtained in step (c).

In another embodiment, the invention further provides a method ofobtaining an electron density map of a ribosomal subunit of interestwhich is related to a ribosomal subunit whose structure is known. Themethod comprises the steps of: (a) producing a crystal of a ribosomalsubunit of interest, wherein the crystal crystallizes in a differentunit cell with different symmetry than the crystal of the ribosomalsubunit whose structure is known; (b) obtaining X-ray diffraction dataof the crystal of interest; (c) inserting the atomic co-ordinates of theknown ribosomal subunit into the unit cell of the crystal of interestand modeling the co-ordinates such that they would be capable ofproducing theoretical X-ray diffraction data that resembles the X-raydiffraction data obtained in step (b); (d) obtaining phases of thecrystal of interest from the modeled co-ordinates in step (c); and (e)obtaining an electron density map of the ribosomal subunit of interestfrom the X-ray diffraction data obtained in step (b) an d the phasesobtained in step (d).

In addition, the invention provides a method of obtaining a model of aribosomal subunit of interest, where the ribosomal subunit of interestdiverges significantly from but is still homologous to the ribosomalsubunit that was used to generate computed phases. The method comprisesthe steps of: (a) providing the atomic co-ordinates of the ribosomalsubunit whose structure is known; and (b) using homology modeling toproduce atomic co-ordinates of the ribosomal subunit of interest.

In another aspect, the invention provides a method of growing a crystalof a ribosome or a ribosomal subunit, as well as crystals resulting fromsuch a method. The method comprises the steps of: (a) isolating aribosome or a ribosomal subunit; (b) precipitating the ribosome orribosomal subunit; (c) back-extracting the precipitated ribosome orribosomal subunit to obtain a solution; (d) seeding the back-extractedsolution; (e) growing a crystal of the ribosome or ribosomal subunitfrom the seeded solution by vapor diffusion at room temperature; and (f)harvesting the crystal. Optionally, the method may further comprise oneor more of the following steps: (g) stabilizing the crystal by gradualtransfer into a solution containing high salt concentration, forexample, from about 1.2 M salt to about 1.7 M salt; (h) maintaining thecrystal under such a high salt concentration; and (i) flash freezing thecrystal.

In another aspect, the invention provides a method of obtaining X-raydiffraction data for a crystal of a ribosome or a ribosomal subunit. Themethod comprises the steps of: (a) obtaining a crystal of a ribosome ora ribosomal subunit, wherein the crystal has one or more of thefollowing characteristics (1) an average thickness of greater than 15μm, and (2) untwinned; and (b) using X-ray crystallography to obtainX-ray diffraction data for the crystal of the ribosome or ribosomalsubunit. The present invention also discloses a method of obtaining anelectron density map of a ribosome or a ribosomal subunit comprisingusing the X-ray diffraction data described herein to obtain an electrondensity map of the ribosome or ribosomal subunit.

In another aspect, the invention provides a method of obtaining X-raydiffraction data for a complex of a ribosome and a ligand, for example,a protein synthesis inhibitor, or a complex of a ribosomal subunit and aligand. The method comprises the steps of (a) obtaining a crystal of aribosome or a ribosomal subunit, wherein the crystal has one or more ofthe following characteristics: (1) an average thickness of greater than15 μm, and (2) untwinned; (b) diffusing a ligand into the crystal andpermitting the ligand to attach to the crystal so as to form a complex;and (c) using X-ray crystallography to obtain X-ray diffraction data forthe complex. In an alternative aspect, the invention provides a methodof obtaining X-ray diffraction data for a complex of a ribosome and aligand, for example, a protein synthesis inhibitor or for a ribosomalsubunit and a ligand. The method comprises the steps of: (a) obtaining aco-crystal for a complex of a ribosome and a ligand or for a complex ofa ribosomal subunit and a ligand, wherein the co-crystal has one or moreof the following characteristics: (1) an average thickness of greaterthan 15 μm, and (2) untwinned; and (b) using X-ray crystallography toobtain X-ray diffraction data for the complex. In either method, theX-ray diffraction data can be used to produce an electron density mapfor a complex of a ribosome and a ligand or for a complex of a ribosomalsubunit and a ligand.

In a preferred embodiment, the invention provides a method of locatingthe attachment of such a ligand to a ribosome or the attachment of theligand to a ribosomal subunit. The method comprises the steps of: (a)obtaining X-ray diffraction data for a ribosome or for a ribosomalsubunit; (b) obtaining X-ray diffraction data for a complex of aribosome and a ligand or for a complex of a ribosomal subunit and aligand; (c) subtracting the X-ray diffraction data obtained in step (a)from the X-ray diffraction data obtained in step (b) to obtain thedifference in the X-ray diffraction data; (d) obtaining phases thatcorrespond to X-ray diffraction data obtained in step (a) using one ormore of the techniques selected from the group consisting of MIR, MIRAS,SAD and computation from an existing atomic structure; (e) utilizing thephases obtained in step (d) and the difference in the X-ray diffractiondata obtained in step (c) to compute a difference Fourier image of theligand; and (f) locating the attachment of the ligand to a ribosome orthe attachment of the ligand to a ribosomal subunit based on thecomputations obtained in step (e).

In another embodiment, the invention provides an alternative method ofobtaining a map of such a ligand attached to a ribosome or of a ligandattached to a ribosomal subunit. The method comprises the steps of: (a)obtaining X-ray diffraction data for a ribosome or for a ribosomalsubunit; (b) obtaining X-ray diffraction data for a complex of aribosome and a ligand or a complex of a ribosomal subunit and a ligand;(c) obtaining phases that correspond to X-ray diffraction data obtainedin step (a) using one or more of the techniques selected from the groupconsisting of MIR, MIRAS, SAD and computation from an existing atomicstructure; and (d) utilizing the phases obtained in step (c) and theX-ray diffraction data obtained in step (b) to compute a map of theligand and the ribosome or of the ligand and the ribosomal subunit.

In another aspect, the invention provides a computer system comprising:(a) a memory having stored therein data indicative of atomicco-ordinates derived from an electron density map having a resolution ofat least about 4.5 Å, more preferably of at least about 3.0 Å, and mostpreferably of about 2.4 Å and defining a ribofunctional locus of a largesubunit of a ribosome; and (b) a processor in electrical communicationwith the memory, the processor comprising a program for generating athree-dimensional model representative of the ribofunctional locus. In apreferred embodiment, the computer system further comprises a device,for example, a computer monitor, or terminal for providing a visualrepresentation of the molecular model. In another preferred embodiment,the processor further comprises one or more programs to facilitaterational drug design.

In a preferred embodiment, the computer system further comprises atleast a portion of the atomic co-ordinates deposited at the Protein DataBank under accession number PDB ID: 1FFK, 1FFZ, 1FG0, or 1JJ2. Inanother preferred embodiment, the atomic co-ordinates further define atleast a portion of a protein synthesis inhibitor, for example, anantibiotic, more specifically an antibiotic selected from the groupconsisting of anisomycin, blasticidin, carbomycin, sparsomycin,spiramycin, tylosin and virginiamycin, complexed with a ribofunctionallocus, for example, at least a portion of the atomic co-ordinatesrecorded on compact disk Disk No. 3 of 3, included herein.

In a preferred embodiment, the ribofunctional locus comprises at least aportion of an active site in the ribosomal subunit, for example, atleast a portion of one or more of: a peptidyl transferase site (aportion of which may be defined by a plurality of residues set forth inTable 5); an A-site (a portion of which may be defined by a plurality ofresidues set forth in Table 6); a P-site (a portion of which may bedefined by a plurality of residues set forth in Table 7); a polypeptideexit tunnel (a portion of which may be defined by a plurality ofresidues set forth in Table 8, Table 9 or Table 10); or an antibioticbinding domain (a portion of which may be defined by a plurality ofresidues set forth in Table 11, Table 12, Table 13, Table 14, Table 15,Table 16 or Table 17). Plurality of residues shall be considered toinclude at least 3 residues, preferably at least 5 residues, and morepreferably at least 10 residues. The ribofunctional locus may be definedby atoms of ribosomal RNA, one or more ribosomal proteins, or acombination of ribosomal RNA and one or more ribosomal proteins.

In another preferred embodiment, the atomic co-ordinates are produced bymolecular modeling. Using the atomic co-ordinates provided herein, theskilled artisan may generate models of any ribosome of interest usingconventional techniques, for example, conventional homology modeling,and or molecular replacement techniques. In another embodiment, theatomic co-ordinates are produced by homology modeling using at least aportion of the atomic co-ordinates deposited at the Protein Data Bankunder accession number PDB ID: 1FFK, 1FFZ, 1FG0, or 1JJ2, or the atomicco-ordinates included in compact disk Disk No. 3 of 3. In anotherembodiment, the atomic co-ordinates are produced by molecularreplacement using at least a portion of the atomic co-ordinatesdeposited at the Protein Data Bank under accession number PDB ID: 1FFK,1FFZ, 1FG0, or 1JJ2, or the atomic co-ordinates included in compact diskDisk No. 3 of 3.

In a preferred embodiment, the atomic co-ordinates define residues thatare conserved between ribosomes or ribosomal subunits of pathogens, forexample, prokaryotic organisms, and, optionally but more preferably, arealso absent from ribosomes or ribosomal subunits of a host organism, forexample, a human. In another preferred embodiment, the atomicco-ordinates may define residues that are conserved between ribosomes orribosomal subunits of prokaryotic organisms, for example, bacteria, and,optionally but more preferably, are also absent from ribosomal subunitsof eukaryotes, for example, a mammal, more preferably, a human. Thisinformation can be used, for example, via the use of one or moremolecular models, to identify targets for rational drug design that maybe exploited to develop new molecules, for example, protein synthesisinhibitors, that disrupt protein synthesis in a pathogen, for example, abacteria, but do not disrupt or otherwise substantially affect proteinsynthesis in a host organism, for example, a human.

In another aspect, the invention provides a variety of methods fordesigning, testing and refining new molecules via rational drug design.For example, the invention provides a method that comprises the stepsof: (a) providing a model, for example, a molecular model, having aribofunctional locus of a large subunit of a ribosome, wherein the modelis defined by the spatial arrangement of atoms derived from an electrondensity map having a resolution of at least about 4.5 Å, more preferablyto at least about 3.0 Å, and most preferably to about 2.4 Å; and (b)using the model to identify a candidate molecule having a surfacecomplementary to the ribofunctional locus. Preferably, the candidatemolecule stereochemically interfits and more preferably binds with theribofunctional locus of the large subunit of the ribosome.

In a preferred embodiment, the method comprises one or more additionalsteps of: producing the candidate molecule identified in such a method;determining whether the candidate molecule, when produced, modulates(for example, induces or reduces) ribosomal activity; identifying amodified molecule; producing the modified molecule; determining whetherthe modified molecule, when produced, modulates ribosomal activity; andproducing the modified molecule for use either alone or in combinationwith a pharmaceutically acceptable carrier. The candidate moleculeand/or the modified molecule may be an antibiotic or antibioticanalogue, for example, a macrolide antibiotic or a macrolide analogue.

In a preferred embodiment, the ribofunctional locus used in such amethod comprises at least a portion of an active site in the ribosomalsubunit. In another preferred embodiment, the ribofunctional locus isdefined by at least a portion of one or more of: a peptidyl transferasesite (a portion of which may be defined by a plurality of residues setforth in Table 5); an A-site (a portion of which may be defined by aplurality of residues set forth in Table 6); a P-site (a portion ofwhich may be defined by a plurality of residues set forth in Table 7); apolypeptide exit tunnel (a portion of which may be defined by aplurality of residues set forth in Table 8, Table 9 or Table 10); or anantibiotic binding domain (a portion of which may be defined by aplurality of residues set forth in Table 11, Table 12, Table 13, Table14, Table 15, Table 16 or Table 17). The ribofunctional locus may bedefined by atoms of ribosomal RNA, one or more ribosomal proteins, or acombination of ribosomal RNA and one or more ribosomal proteins.

In another preferred embodiment, the atomic co-ordinates are used toproduce a molecular model in an electronic form. The atomic co-ordinatespreferably are produced by molecular modeling. In another embodiment,the atomic co-ordinates are produced by homology modeling using at leasta portion of the atomic co-ordinates deposited at the Protein Data Bankunder accession number PDB ID: 1FFK, 1FFZ, 1FG0, or 1JJ2, or the atomicco-ordinates included in compact disk Disk No. 3 of 3. In anotherembodiment, the atomic co-ordinates are produced by molecularreplacement using at least a portion of the atomic co-ordinatesdeposited at the Protein Data Bank under accession number PDB ID: 1FFK,1FFZ, 1FG0, or 1JJ2, or the atomic co-ordinates included in compact diskDisk No. 3 of 3.

In a preferred embodiment, the atomic co-ordinates may define residuesthat are conserved among ribosomes or ribosomal subunits of pathogens,for example, prokaryotic organisms, and, optionally but more preferably,are also absent in ribosomes or ribosomal subunits of a host organism,for example, a human. In another preferred embodiment, the atomicco-ordinates may define residues that are conserved between ribosomes orribosomal subunits of prokaryotic organisms, for example, bacteria, and,optionally but more preferably, are also absent from ribosomes orribosomal subunits of eukaryotes, for example, a mammal, more preferablya human. This information can be used, for example, via the use of oneor more molecular models, to identify targets for rational drug designthat may be exploited to develop new molecules, for example, proteinsynthesis inhibitors, that disrupt protein synthesis in a pathogen, forexample, a bacteria but do not disrupt or otherwise substantially affectprotein synthesis in a host organism, for example, a human.

In a preferred embodiment, the invention provides a method of obtaininga modified agent. The method comprises the steps of: (a) obtaining acrystal of a ribosome or of a ribosomal subunit; (b) obtaining theatomic co-ordinates of the crystal; (c) using the atomic co-ordinatesand one or more molecular modeling techniques, for example, graphicmolecular modeling and computational chemistry, to determine how tomodify the interaction of an agent with a ribosome or ribosomal subunit;and (d) modifying the agent based on the determinations obtained in step(c) to produce a modified agent. Alternatively, the method furthercomprises contacting the modified agent with a ribosome or ribosomalsubunit and detecting the interaction of the agent to the ribosome orribosomal subunit. The present invention also provides such a modifiedagent (preferably a therapeutic agent), wherein the modified agent bindsdifferently to a ribosome or ribosomal subunit than does the agent fromwhich the modified agent was derived.

In another aspect, the invention provides new protein synthesisinhibitors that disrupt the function of a target ribosome. Theseinhibitors can be readily designed and tested as disclosed herein.

One type of protein synthesis inhibitor of the invention comprises: afirst binding domain having a surface, for example, a solvent accessiblesurface, that mimics or duplicates a surface of a known first molecule,for example, a first antibiotic, that binds with a first contact site,for example, a first ribofunctional locus, in or on a large ribosomalsubunit; and a second binding domain having a surface, for example, asolvent accessible surface, that mimics or duplicates a surface of aknown second molecule, for example, a second antibiotic, that binds witha second contact site, for example, a second ribofunctional locus, in oron the ribosomal subunit. The first domain is attached to the seconddomain so as to permit both the first domain and the second domain tobind simultaneously with their respective contact sites within or on theribosomal subunit so as to disrupt protein synthesis in a ribosomalsubunit.

Another type of protein synthesis inhibitor is a synthetic, engineeredmolecule that comprises: a binding domain having a surface, for example,a solvent accessible surface, that mimics or duplicates a solventaccessible surface of a known molecule, for example, a first knownantibiotic, which binds with a contact site, for example, aribofunctional locus in or on a ribosomal subunit; and an effectordomain attached to the binding domain which, upon binding of the bindingdomain with the contact site, occupies a space within or adjacent theribosomal subunit thereby to disrupt protein synthesis in the ribosomalsubunit.

The foregoing aspects and embodiments of the invention may be more fullyunderstood by reference to the following figures, detailed descriptionand claims. Further advantages are evident from the drawings (providedin both grayscale and color).

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Color renditions similar to many of the following figures can be found,for example, in Ban et al. (2000) Science 289: 905-920; or Nissen et al.(2000) Science 289: 920-929.

The objects and features of the invention may be more fully understoodby reference to the drawings described below:

FIGS. 1(A)-(E) show the electron density from a 2.4 Å resolutionelectron density map. Specifically, FIG. 1(A) shows a stereo view of ajunction between 23S rRNA domains II, III, and IV. FIG. 1(B) shows theextended region of protein L2 interacting with surrounding RNA. FIG.1(C) shows in detail the L2 region with a bound Mg²⁺ ion. FIG. 1(D)shows in detail L2 with amino acid side chains. FIG. 1(E) shows helices94-97 from domain 6.

FIG. 2 shows the H. marismortui large ribosomal subunit in the crownview. The subunit is shown in the crown view, with its L7/L12 stalk tothe right, its L1 stalk to the left, and its central protuberance (CP)up. In this view, the surface of the subunit that interacts with thesmall ribosomal subunit faces the reader. RNA is shown in gray in aspace-filling rendering. The backbones of the proteins visible arerendered in gold. A transition state analogue bound to the peptidyltransferase site of the subunit is indicated in green. The particle isapproximately 250 Å across.

FIGS. 3(A)-(B) show the secondary structure of the 23S rRNA from H.marismortui. The secondary structure of this 23S rRNA is shown in astandardized format. FIG. 3(A) shows the 5′ half of the large subunitrRNA. FIG. 3(B) show the 3′ half of the large subunit rRNA. This diagramshows all the base pairings seen in the crystal structure of the largesubunit that are stabilized by at least two hydrogen bonds. Pairingsshown in red were predicted and are observed. Those shown in green werepredicted, but are not observed. Interactions shown in blue areobserved, but were not predicted. Bases shown in black do not appear tobe involved in pairing interactions. Sequences that cannot be visualizedin the 2.4 Å resolution electron density map are depicted in gray withthe secondary structures predicted for them.

FIGS. 4(A)-(L) show the tertiary structures of the RNA domains in the H.marismortui large ribosomal subunit, its RNA as a whole, and schematicsof its RNAs. Specifically, FIGS. 4(A) and 4(B) show the RNA structure ofthe entire subunit. Domains are color coded as shown in the schematic ofFIG. 5(C). FIG. 4(A) shows the particle in the crown view. FIG. 4(B)shows the image in FIG. 4(A) rotated 180° about an axis runningvertically in the plane of the image. FIGS. 4(C) and 4(D) show aschematic diagram of 23S rRNA and the secondary structure of 5S rRNA.FIG. 4(C) shows a schematic diagram of 23S rRNA secondary structure ofFIG. 3 with helices numbered according to Leffers et al. ((1987) J. Mol.Biol. 195: 43-61), and the domains of the molecule are indicated bycolor shading. FIG. 4(D) shows the secondary structure of 5S rRNA fromH. marismortui. Thick lines joining bases represent Watson-Crickpairing. Bases joined by a lower case “o” indicate non-Watson-Crickpairing. Bases joined by thin lines interact via a single hydrogen bond.Bases shown in black are not paired. Bases shown in red arephylogenetically predicted pairing that have now been confirmed(Symanski et al. (1998) Nucl. Acids Res. 26: 156-159). Pairs shown inblue are observed, but were not predicted, and pairs shown in green werepredicted but are not observed. FIGS. 4(E) through 4(L) show stereoviews of the RNA domains in the 23S rRNA and of 5S rRNA. Each domain iscolor-coded from its 5′ end to its 3′ end to facilitate the viewerfollowing its trajectory in three-dimensions. The surfaces where themost important inter-domain interactions occur are shown in mono to theright of the stereo views. FIG. 4(E) shows domain I; FIG. 4(F) showsdomain II; FIG. 4(G) shows domain III; FIG. 4(H) shows domain IV; FIG.4(I) shows domain V, crown view; FIG. 4(J) shows domain V, back view;FIG. 4(K) shows domain VI; and FIG. 4(L) shows 5S rRNA.

FIGS. 5(A)-(C) show conservations and expansions in the 23S rRNA of H.marismortui. The generality of the RNA in these images is gray.Sequences that are found to be >95% conserved across the threephylogenetic kingdoms are shown in red. Sequences where expansion in thebasic 23S structure is permitted are shown in green (Gutell et al.(2000) supra). Specifically, FIG. 5(A) shows the particle rotated withrespect to the crown view so that its active site cleft can be seen.FIG. 5(B) shows the crown view. FIG. 5(C) shows the back view of theparticle, i.e., the crown view rotated 180° about its vertical axis.

FIGS. 6(A)-(I) show structures of some large subunit ribosomal proteinsthat have non-globular extensions. Only the backbones of the proteinsare shown. The globular domains of these proteins are shown in green,and their non-globular extensions are depicted in red. The positions ofthe zinc ions in L44e and L37e are also indicated. FIG. 6(A) shows L2;FIG. 6(B) shows L3; FIG. 6(C) shows L39; FIG. 6(D) shows L4; FIG. 6(E)shows L15; FIG. 6(F) shows L21e; FIG. 6(G) shows L44e; FIG. 6(H) showsL37e; FIG. 6(I) shows L19;

FIGS. 7(A)-(C) show proteins that appear on the surface of the largeribosomal subunit. The RNA of the subunit is shown in gray, as in FIG.2, and protein backbones are shown in gold. Specifically, FIG. 7(A)shows the subunit in the crown view of the subunit. FIG. 7(B) shows theback side of the subunit in the crown view orientation. FIG. 7(C) showsthe bottom view; the end of the peptide tunnel appears in the center ofthis image. The proteins visible in each image are identified in thesmall images at the lower left corner of the Figure.

FIGS. 8(A)-(F) show the protein distribution and protein-RNAinteractions in the large ribosomal subunit. Specifically, FIG. 8(A)shows the structures of proteins in the neighborhood of the end of thepeptide tunnel and how they relate to the RNA sequences with which theyinteract. Protein L22 extends a long β hairpin extension inside the 23SrRNA. L24 has a similar extension but the entire protein is on thesurface of the particle. L39 is the only protein in the subunit thatlacks tertiary structure, while L37e has both NH₂ and COOH terminalextensions. L19 is unique in having two globular domains on the surfaceof the subunit connected by an extended sequence that weaves through theRNA. The end of L39 (green) actually enters the tunnel, while L37e (red)is entirely surrounded by RNA. FIG. 8(B) shows the non-globularextensions of L2 and L3 reaching through the mass of 23S rRNA towardsthe peptidyl transferase site, which is marked by a CCdA-p-puromycinmolecule. FIG. 8(C) shows L22 interacting with portions of all six ofthe domains of 23S rRNA. FIG. 8(D) shows a schematic of 23S rRNA showingthe locations of the sequences that make at least van der Waals contactwith protein (red). FIG. 8(E) shows a stereo view of the proteins of thelarge ribosomal subunit with all the RNA stripped away. Proteins arecolor red as an aid to visualization only. FIG. 8(F) shows a crosssection of the subunit in the area of the tunnel exit. Protein L22 isshown as ribbons in red, and the P hairpin loop where mutations confererythromycin resistance is shown in orange. Atoms on the surface areshown in gray, protein atoms are shown in green, and atoms at the sliceinterface are shown in blue.

FIGS. 9(A)-(C) show chemical structures of ribosome peptidyl transferasesubstrates and analogues. Specifically, FIG. 9(A) shows the tetrahedralcarbon intermediate produced during peptide bond formation; thetetrahedral carbon is indicated by an arrow. FIG. 9(B) shows thetransition state analogue formed by coupling the 3′ OH of CCdA to theamino group of the O-methyl tyrosine residue of puromycin via aphosphate group, CCdA-p-Puro (Welch et al. (1995) supra). FIG. 9(C)shows an amino-N-acylated mini helix constructed to target the A-site.The oligonucleotide sequence 5′ phosphateCCGGCGGGCUGGUUCAAACCGGCCCGCCGGACC 3′ (SEQ ID NO: 1) puromycin shouldform 12 base pairs. The construct was based on a mini helix which is asuitable substrate for amino-acylation by Tyr-tRNA synthetase. The 3′ OHof its terminal C is coupled to the 5′ OH of the N6-dimethyl A moiety ofpuromycin by a phosphodiester bond.

FIGS. 10(A)-(C) show experimentally phased electron density maps of thesubstrate analogue complexes at 3.2 Å resolution, with modelssuperimposed (oxygen, red; phosphorus, purple; nitrogen, blue; andcarbon, green for rRNA and yellow for substrate). Specifically, FIG.10(A) shows an F_(o)(complex)-F_(o)(parent) difference electron densitymap with a skeletal model of CCdA-p-Puro superimposed. FIG. 10(B) showsa 2F_(o)(complex)-Fb(parent) electron-density map of the CCdA-p-Puro inthe active site region with the structures of the ribosome and inhibitorsuperimposed showing the proximity of the N3 of A2486 (2451) to thephosphate, non-bridging oxygen in this complex. FIG. 10(C) shows anF_(o)(complex)-F_(o)(parent) differences electron density map of thetRNA acceptor stem analogue with a skeletal model of CCpurosuperimposed. There is density only for the ribose and phosphate of C74and none for the rest of the RNA hairpin.

FIGS. 11(A) and (B) show a combined model of the CCA portion of the minihelix bound to the A-site and CCdA-p-Puro bound to the A- and P-sites,color coded as in FIG. 2. Specifically, FIG. 11(A) shows thebase-pairing interactions between the P-site C74 and C75 and the P loopof 23S rRNA on the left and the A-site C75 with the A loop of 23S rRNAon the right. The catalytic A2486 is near the phosphate oxygen (P) thatis the analogue of the tetrahedral intermediate oxyanion. FIG. 11(B)shows A2637 (in all blue) lying between the two CCA's and A2486 (green)whose N3 approaches a non-bridging phosphate oxygen. The N1 atoms of theA76 bases from the A- and P-site tRNAs are making nearly identicalinteractions with a ribose 2′ OH in both the A- and P-loops,respectively, and an approximate 2-fold axis relates these residues.

FIG. 12 shows a space filling model of the 23S and 5S rRNA, the proteinsand the CCdA-p-Puro inhibitor viewed down the active site cleft in arotated “crown view.” The bases are white and the sugar phosphatebackbones are yellow. The inhibitor is shown in red and the numberedproteins are shown in blue. The L1 and L11 proteins positioned at lowerresolution are in blue backbone. The central protuberance is labeled CP.

FIG. 13(A) shows a stereo view diagram of the three-dimensionaldistribution of the residues comprising the loops A and P and thepeptidyl transferase loop. FIG. 13(B) shows a stereo view of the centralloop in domain V from the direction of the tunnel. The residues arecolor coded based on mutations which confer antibiotic resistance. FIG.13(C) shows domain V active site with its central loop shown as thesecondary structure.

FIGS. 14(A) and (B) show the closest approach of polypeptides to thepeptidyl transferase active site marked by a ball and stickrepresentation of the Yarus inhibitor, CCdA-p-Puro. Specifically, FIG.14(A) shows a coil representation of domain V RNA backbone in red andbases in gray and a ribbon backbone representation of all thirteenproteins that interact with it. FIG. 14(B) shows a close-up view of theactive site with the RNA removed. The phosphate of the Yarus analogueand the proteins whose extensions are closest to the inhibitor are shownin ribbon with their closest side-chains in all atom representation. Thedistances in A between the closest protein atoms and the phosphorousanalogue of the tetrahedral carbon (pink) are shown, as is a modeledpeptide (pink).

FIG. 15 shows conserved nucleotides in the peptidyl transferase regionthat binds CCdA-p-Puro A space filling representation of the active siteregion with the Yarus inhibitor viewed down the active site cleft. Allatoms belonging to 23S rRNA nucleotides that are 95% conserved in allthree kingdoms (Gutell et al. (2000) supra) are colored red and allother nucleotides are white; the inhibitor is colored blue.

FIGS. 16(A)-(C) show the catalytic apparatus of the peptidyl transferaseactive site. Specifically, FIG. 16(A) shows stereo view of a portion ofthe experimental 2.4 Å resolution electron density map (Ban et al.(2000) Science 289: 905-920) of the large subunit in the region of thecatalytic site in stereo. The structure the RNA involved in interactionswith A2486 is superimposed. Residues G2102 (2061) and G2482 (2447) arehydrogen bonded to the N6 of A2486 (2451) and G2482 which interacts witha neighboring phosphate group. FIG. 16(B) shows a skeletalrepresentation with dashed hydrogen-bonds showing G2482, G2102, A2486and the buried phosphate that is proposed to result in a charge relaythrough G2482 to the N3 of A2486. FIG. 16(C) shows the normal and rarerimine tautomeric forms of G2482 and A2486 that are proposed to bestabilized by the buried phosphate of residue 2485.

FIGS. 17(A)-(C) show the proposed mechanism of peptide synthesiscatalyzed by the ribosome. Specifically, FIG. 17(A) shows the N3 ofA2486 abstracting a proton from the NH₂ group as the latter attacks thecarbonyl carbon of the peptidyl-tRNA. FIG. 17(B) shows a protonated N3stabilizing the tetrahedral carbon intermediate by hydrogen bonding tothe oxyanion. FIG. 17(C) shows the proton transferred from the N3 to thepeptidyl tRNA 3′ OH as the newly formed peptide deacylates.

FIGS. 18(A) and (B) show space filling representations of the 50Sribosomal subunit with the 3 tRNA molecules, in the same relativeorientation that they are found in the 70S ribosome structure by Nollerand colleagues docked onto the CCA's bound in the A-Site and P-Site.Specifically, FIG. 18(A), shown on the left-hand side, shows the wholesubunit in rotated crown view with the rRNA in yellow, proteins in pinkand tRNAs in orange. FIG. 18(B), shown on the right-hand side, shows aclose-up view showing the numbered proteins are in pink and the rRNA inblue. A backbone ribbon representation of the A-, P-, and E-sites areshown in yellow, red and white, respectively.

FIGS. 19(A)-(F) show the polypeptide exit tunnel. Specifically, FIG.19(A) shows the subunit cut in half, roughly bisecting its centralprotuberance and its peptide tunnel along the entire length. The twohalves have been opened like the pages of a book. All ribosome atoms areshown in CPK representation, with all RNA atoms that do not contactsolvent shown in white and all protein atoms that do not contact solventshown in green. Surface atoms of both protein and RNA are color-codedwith carbon in yellow, oxygen in red, and nitrogen in blue. A possibletrajectory for a polypeptide passing through the tunnel is shown as awhite ribbon. The peptidyl transferase site (PT) is also shown. FIG.19(B) shows detail of the polypeptide exit tunnel with the distributionof polar and non-polar groups, with atoms colored as in FIG. 19(A), theconstriction in the tunnel formed by proteins L22 and L4 (green patchesclose to PT), and the relatively wide exit of the tunnel. A modeledpolypeptide is in white. FIG. 19(C) shows the tunnel surface withbackbone atoms of the RNA color coded by domain: domain I (white), II(light blue), III (gold), IV (green), V (orange), 5S (pink) and proteinsare blue. The peptidyl transferase center (PTC) is shown. FIG. 19(D) isa space filling representation of the large subunit surface at thetunnel exit showing the arrangement of proteins, some of which mightplay roles in protein secretion. The RNA is in white (bases) and yellow(backbone) and the numbered proteins are blue. A modeled polypeptide isexiting the tunnel in red. FIG. 19(E) shows a close-up view of the halfof the exit tunnel showing the relationship of the peptidyl transferasecenter (PTC) to proteins L4 (yellow) and L22 (blue). The Yarus inhibitorand a modeled peptide are purple and the 23S rRNA is in red and white.FIG. 19(F) shows a secondary structure schematic of 23S rRNA identifyingthe sequences that contact the tunnel in red.

FIG. 20 is a picture showing the spatial relationship between theantibiotic anisomycin bound to a large ribosomal subunit.

FIG. 21 is a picture showing the spatial relationship between theantibiotic blasticidin bound to a large ribosomal subunit.

FIG. 22 is a picture showing the spatial relationship between theantibiotics carbomycin and tylosin bound to a large ribosomal subunit.

FIG. 23 is a picture showing the spatial relationship between theantibiotic sparsomycin bound to a large ribosomal subunit.

FIG. 24 is a picture showing the spatial relationship between theantibiotics virginiamycin (streptogramin A) and carbomycin bound to alarge ribosomal subunit.

FIG. 25 is a picture showing the spatial relationship of certainantibiotics, namely, anisomycin, blasticidin, carbomycin, andvirginiamycin, bound to a large ribosomal subunit. The locations of thebound antibiotics are shown relative to the ribosomal A-site, P-site,and polypeptide exit tunnel.

FIGS. 26(A)-(C) are pictures showing a peptidyl transferase sitedisposed within a large ribosomal subunit. FIG. 26A shows a boundtylosin molecule, and identifies a disaccharide binding pocket and twocavities denoted “cavity 1” and “cavity 2.” FIGS. 26(B) and (C) areprovided on the left hand side to orient the reader to the locations ofthe peptidyl transferase site (PT) and polypeptide exit tunnel in thelarge ribosomal subunit.

FIG. 27 is a schematic representation of a computer system useful inmolecular modeling a ribosomal subunit and/or for performing rationaldrug design.

FIG. 28 is a schematic representation of certain potential drug targetsites in a large ribosomal subunit.

FIGS. 29(A)-(D) are pictures showing the residues within the wall of thepolypeptide exit tunnel that are conserved (red) or non-conserved (blue)between E. coli and rat. The ribosomal subunit has been sliced down thepolypeptide exit tunnel with one half of the polypeptide exit tunnelshown in FIG. 29(A), and the other half of the polypeptide exit tunnelis shown in FIG. 29(B). FIG. 29(C) is provided to orient the reader toshow the location of the portion of the ribosomal subunit shown in FIG.29(A) relative to the ribosomal subunit as a whole. FIG. 29(D) isprovided to orient the reader to show the location of the portion of theribosomal subunit shown in FIG. 29(B) relative to the large ribosomalsubunit as a whole.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

As used herein, the term “active site” refers to regions on a ribosomeor ribosomal subunit that are directly involved in protein synthesis,e.g., the peptidyl transferase site, the elongation factor binding site,and other similar sites.

As used herein, the terms “agent” and “ligand” are used synonymously andrefer to any atom, molecule, or chemical group which binds with aribosome, ribosomal subunit or ribosome fragment. Thus, ligands include,but are not limited to, a single heavy atom, an antibiotic, a tRNA, apeptidyl tRNA, an aminoacyl tRNA, or a signal recognition particle(“SRP”).

As used herein, “archaebacteria” refers to the kingdom of monerans thatincludes methane producers, sulfur-dependent species, and many speciesthat tolerate very salty or hot environments.

As used herein, the term “A-site” refers to the locus occupied by anaminoacyl-tRNA molecule immediately prior to its participation in thepeptide-bond forming reaction.

As used herein, the term “asymmetric unit” refers to a minimal set ofatomic co-ordinates that when operated upon by the symmetry operationsof a crystal will regenerate the entire crystal.

As used herein, “at least a portion of” or “at least a portion of thethree-dimensional structure of” is understood to mean a portion of thethree-dimensional structure of a ribosome or ribosomal subunit,including charge distribution and hydrophilicity/hydrophobicitycharacteristics, formed by at least three, more preferably at leastthree to ten, and most preferably at least ten contiguous amino acidand/or nucleotide residues of the ribosome or ribosomal subunit. Thecontiguous residues forming such a portion may be residues which form acontiguous portion of the primary sequence of a ribosomal RNA orribosomal protein, residues which form a contiguous portion of thethree-dimensional structure of the ribosome or ribosomal subunit, or acombination thereof. Thus, the residues forming a portion of thethree-dimensional structure need not be contiguous in the primarysequence but, rather, must be contiguous in space. As used herein, theresidues forming “a portion of the three-dimensional structure of” aribosome or ribosomal subunit, form a contiguous three-dimensional shapein which each atom or functional group forming the portion of the shapeis separated from the nearest atom or functional group forming theportion of the shape by no more than 40 Å, preferably by no more than 20Å, more preferably by no more than 5-10 Å, and most preferably by nomore than 1-5 Å.

As used herein, the term “atomic co-ordinates” or “structureco-ordinates” refers to mathematical co-ordinates (represented as “X,”“Y” and “Z” values) that describe the positions of atoms in a crystal ofa ribosome or ribosomal subunit. The diffraction data obtained from thecrystals are used to calculate an electron density map of the repeatingunit of the crystal. The electron density maps are used to establish thepositions of the individual atoms within a single ribosomal subunit.Those of skill in the art understand that a set of structureco-ordinates determined by X-ray crystallography is not without standarderror. For the purpose of this invention, any set of structureco-ordinates for a ribosome or ribosomal subunit from any source has aroot mean square deviation of non-hydrogen atoms of less than 0.75 Åwhen superimposed on the non-hydrogen atom positions of the said atomicco-ordinates deposited at the Research Collaboratory for StructuralBioinformatics (RCSB) Protein Data Bank (PDB) (Berman et al. (2000)Nucleic Acids Research 28, 235-242; http://www.rcsb.org/pdb/) with theaccession numbers PDB ID: 1FFK; PDB ID: 1FFZ; PDB ID: 1FG0; or PDB ID:1JJ2, the disclosure of each of the foregoing of which is incorporatedherein by reference in its entirety.

In the list of atomic co-ordinates deposited at the RCSB Protein DataBank or included herein as files recorded on the compact disks, the term“atomic co-ordinate” or structure co-ordinates refer to the measuredposition of an atom in the structure in Protein Data Bank (PDB) format,including X, Y, Z and B, for each. The term “atom type” refers to theelement whose co-ordinates are measured. The first letter in the columndefines the element. The term “X”, “Y”, “Z” refers to thecrystallographically defined atomic position of the element measuredwith respect to the chosen crystallographic origin. The term “B” refersto a thermal factor that measures the mean variation of an atom'sposition with respect to its average position.

Reference is made to the sets of atomic co-ordinates and related tablesincluded with this specification and submitted on compact disk (sixtotal compact disks including three original compact disks, and aduplicative copy of each of the original compact disks), all of theforegoing of which are incorporated by reference herein. Disk No. 1 of 3contains eight files; Disk No. 2 of 3 contains four files; and Disk No.3 of 3 contains nine files. Disk No. 1 of 3 contains the filesidentified as PDB1FFK.DOC and PDB1FFK.ENT which represent files ofco-ordinates defining the large ribosomal subunit; PDB1FFZ.DOC andPDB1FFZ.ENT which represent files of the co-co-ordinates defining thelarge ribosomal subunit—CCdA-p-Puro complex; and PDB1FGO.DOC andPDB1FGO.ENT which represent files of the co-ordinates defining the largeribosomal subunit—aa-tRNA analogue complex. Disk No. 2 of 3 containsfiles identified as 1JJ2.RTF and 1JJ2.TXT which represent files of theco-ordinates defining the completely refined large ribosomal subunit.Disk No. 3 of 3 contains the files identified as anisomycin.pdb,blasticidin.pdb, carbomycin.pdb, sparsomycin.pdb, spiramycin.pdb,tylosin.pdb and virginiamycin.pdb which represent files of theco-ordinates defining the large ribosomal subunit bound to anisomycin,blasticidin, carbomycin, sparsomycin, spiramycin, tylosin, andvirginiamycin, respectively.

As will be apparent to those of ordinary skill in the art, the atomicstructures presented herein are independent of their orientation, andthat the atomic co-ordinates identified herein merely represent onepossible orientation of a particular large ribosomal subunit. It isapparent, therefore, that the atomic co-ordinates identified herein maybe mathematically rotated, translated, scaled, or a combination thereof,without changing the relative positions of atoms or features of therespective structure. Such mathematical manipulations are intended to beembraced herein.

As used herein, the terms “atomic co-ordinates derived from” and “atomsderived from” refers to atomic co-ordinates or atoms derived, eitherdirectly or indirectly, from an electron density map. It is understoodthat atomic co-ordinates or atoms derived “directly” from an electrondensity map refers to atomic co-ordinates or atoms that are identifiedfrom and/or fitted to an electron density map by using conventionalcrystallographic and/or molecular modeling techniques and thus can beconsidered to be primary atomic co-ordinates or atoms. It is understoodthat atomic co-ordinates or atoms derived “indirectly” from an electrondensity map refers to atomic co-ordinates or atoms that are derived fromand thus are derivatives or transforms of the primary atomicco-ordinates or atoms and thus can be considered to be secondary atomicco-ordinates or atoms. The secondary atomic co-ordinates or atoms may begenerated from the primary atomic co-ordinates or atoms by usingconventional molecular modeling techniques. By way of a non limitingexample, the atomic co-ordinates for the H. marismortui large ribosomalsubunit as described hereinbelow are considered to be primaryco-ordinates, whereas the atomic co-ordinates of a mammalian largeribosomal subunit which can be derived from H. marismortui atomicco-ordinates by molecular modeling, including, for example, homologymodeling and/or molecular replacement, are considered to be secondaryco-ordinates. Both types of atomic co-ordinates and atoms are consideredto be embraced by the invention.

As used herein the terms “bind,” “binding,” “bound,” “bond,” or“bonded,” when used in reference to the association of atoms, molecules,or chemical groups, refer to any physical contact or association of twoor more atoms, molecules, or chemical groups (e.g., the binding of aligand with a ribosomal subunit refers to the physical contact betweenthe ligand and the ribosomal subunit). Such contacts and associationsinclude covalent and non-covalent types of interactions.

As used herein, the terms “complex” or “complexed” refer to the assemblyof two or more molecules to yield a higher order structure, such as, a50S ribosomal subunit bound to a ligand.

As used herein, the term “computational chemistry” refers tocalculations of the physical and chemical properties of the molecules.

As used herein, the term “conjugated system” refers to more than twodouble bonds that are positioned spatially so that their electrons arecompletely delocalized with the entire system. Aromatic residues containconjugated double bond systems.

As used herein, the terms “covalent bond” or “valence bond” refer to achemical bond between two atoms in a molecule created by the sharing ofelectrons, usually in pairs, by the bonded atoms.

As used herein, the term “crystal” refers to any three-dimensionalordered array of molecules that diffracts X-rays.

As used herein, the term “crystallographic origin” refers to a referencepoint in the unit cell with respect to the crystallographic symmetryoperation.

As used herein, the term “elongation factor binding domain” refers tothe region of the ribosome that interacts directly with elongationfactors, including, for example, the elongation factors, EF-Tu and EF-G.

As used herein, the term “E-site” refers to the locus occupied by adeacylated tRNA molecule as it leaves the ribosome following itsparticipation in peptide-bond formation.

As used herein, the term “heavy atom derivatization” refers to themethod of producing a chemically modified form, also known as a “heavyatom derivative”, of a crystal of the ribosome and the ribosomal subunitand its complexes. In practice, a crystal is soaked in a solutioncontaining heavy metal atom salts, or organometallic compounds, e.g.,mercury chlorides, ethyl-mercury phosphate, osmium pentamine, or iridiumpentamine, which can diffuse through the crystal and bind to theribosome or ribosomal subunit. The location(s) of the bound heavy metalatom(s) can be determined by X-ray diffraction analysis of the soakedcrystal. This information, in turn, is used to generate the phaseinformation used to construct three-dimensional structure of the complex(Blundell et al. (1976) supra).

As used herein, the term “homologue” is understood to mean any one orcombination of (i) any protein isolated or isolatable from a ribosome ora ribosomal subunit (i.e., a ribosomal protein), (ii) any nucleic acidsequence isolated or isolatable from a ribosome or ribosomal subunit(i.e., a ribosomal RNA), (iii) any protein having at least 25% sequenceidentity to a ribosomal protein isolated from E. coli or Rattusnorvegicus as determined using the computer program “BLAST” versionnumber 2.1.1 implementing all default parameters, or (iv) any nucleicacid having at least 30% sequence identity to a ribosomal RNA isolatedfrom E. coli or Rattus norvegicus as determined using the computerprogram “BLAST” version number 2.1.1 implementing all defaultparameters. “BLAST” version number 2.1.1 is available and accessible viathe world wide web at http://www/ncbi.nlm.nih.gov/BLAST/ or can be runlocally as a fully executable program on a standalone computer.

As used herein, the term “homology modeling” refers to the practice ofderiving models for three-dimensional structures of macromolecules fromexisting three-dimensional structures for their homologues. Homologymodels are obtained using computer programs that make it possible toalter the identity of residues at positions where the sequence of themolecule of interest is not the same as that of the molecule of knownstructure.

As used herein, the term “hydrogen bond” refers to two electronegativeatoms (either O or N), which share a hydrogen that is covalently bondedto only one atom, while interacting with the other.

As used herein, the term “hydrophobic interaction” refers tointeractions made by two hydrophobic residues.

As referred to herein, ribosomal proteins are designated “LX” or “SX”,where L stands for “large subunit; S stands for “small subunit”; and Xin either case is an integer.

As used herein, the term “MIR” refers to multiple isomorphousreplacement, a technique used for deriving phase information fromcrystals treated with heavy atom compounds.

As used herein, the term “molecular graphics” refers tothree-dimensional representations of atoms, preferably on a computerscreen.

As used herein, the terms “molecular model” or “molecular structure”refer to the three-dimensional arrangement of atoms within a particularobject (e.g., the three-dimensional structure of the atoms that comprisea ribosome or ribosomal subunit, and the atoms that comprise a ligandthat interacts with a ribosome or ribosomal subunit, particularly with alarge ribosomal subunit, more particularly with a 50S ribosomalsubunit).

As used herein, the term “molecular modeling” refers to a method orprocedure that can be performed with or without a computer to make oneor more models, and, optionally, to make predictions about structureactivity relationships of ligands. The methods used in molecularmodeling range from molecular graphics to computational chemistry.

As used herein, the term “molecular replacement” refers to a method thatinvolves generating a model of a ribosome or ribosomal subunit whoseatomic co-ordinates are unknown, by orienting and positioning the atomicco-ordinates described in the present invention in the unit cell of thecrystals of the unknown ribosome so as best to account for the observeddiffraction pattern of the unknown crystal. Phases can then becalculated from this model and combined with the observed amplitudes togive the atomic co-ordinates of the unknown ribosome or ribosomalsubunit. This type of method is described, for example, in The MolecularReplacement Method, (Rossmann, M. G., ed.), Gordon & Breach, New York,(1972).

As used herein, “noncovalent bond” refers to an interaction betweenatoms and/or molecules that does not involve the formation of a covalentbond between them.

As used herein, the term “peptidyl transferase site” refers to the locusin the large ribosomal subunit where peptide bonds are synthesized.

As used herein, the term “polypeptide exit tunnel” refers to the channelthat passes through the large ribosomal subunit from the peptidyltransferase site to the exterior of the ribosome through which newlysynthesized polypeptides pass.

As used herein, the term “protein synthesis inhibitor” refers to anymolecule that can reduce, inhibit or otherwise disrupt protein orpolypeptide synthesis in a ribosome.

As used herein, the term “P-site” refers to the locus occupied by apeptidyl-tRNA at the time it participates in the peptide-bond formingreaction.

As used herein, the term “ribofunctional locus” refers to a region ofthe ribosome or ribosomal subunit that participates, either actively orpassively, in protein or polypeptide synthesis within the ribosome orribosomal subunit and/or export or translocation of a protein orpolypeptide out of a ribosome. The ribofunctional locus can include, forexample, a portion of a peptidyl transferase site, an A-site, a P-site,an E-site, an elongation factor binding domain, a polypeptide exittunnel, and a signal recognition particle (SRP) binding domain. It isunderstood that the ribofunctional locus will not only have a certaintopology but also a particular surface chemistry defined by atoms that,for example, participate in hydrogen bonding (for example, proton donorsand/or acceptors), have specific electrostatic properties and/orhydrophilic or hydrophobic character.

As used herein, the term “ribosomal subunit” refers to one of the twosubunits of the ribosome that can function independently during theinitiation phase of protein synthesis but which both together constitutea ribosome. For example, a prokaryotic ribosome comprises a 50S subunit(large subunit) and a 30S subunit (a small subunit).

As used herein, the term “ribosome” refers to a complex comprising alarge ribosomal subunit and small ribosomal subunit.

As used herein, the term “signal recognition particle binding domain”refers to the portion of the ribosome that interacts directly with thesignal recognition particle.

As used herein, the term “space group” refers to the arrangement ofsymmetry elements of a crystal.

As used herein, the term “symmetry operation” refers to an operation inthe given space group that places the atoms in one asymmetric unit onthe corresponding atoms in another asymmetric unit.

As used herein, the term “twinned” refers to a single macroscopiccrystal that contains microscopic domains of the same symmetry thatdiffer significantly in orientation in such a way that the diffractionpatterns of all are superimposed. In a twinned crystal the mosaicblocks, or domains, are orientated so that some point in one directionand others point in a second, distinctly different direction, and thedirections are such that the diffraction pattern generated by one groupof blocks falls exactly on top of the diffraction pattern of the othergroup.

As used herein, the term “untwinned” refers to a crystal cell thedomains of which are aligned. The domains are also known as the “mosaicblocks.” Most crystals diffract as though they were assemblies of mosaicblocks. One can think of them as small, perfectly ordered regions withinthe larger crystal, which, overall, is not so well ordered. Each blockhas the same symmetry and unit cell packing as all the others.

As used herein, the term “unit cell” refers to a basic parallelepipedshaped block. The entire volume of crystal may be constructed by regularassembly of such blocks. Each unit cell comprises a completerepresentation of the unit of pattern, the repetition of which builds upthe crystal.

II. Structure and Use of the Large Ribosomal Subunit

A. Atomic Structure of the Large Ribosomal

Subunit at 2.4 Å Resolution, Initial Refinement

The present invention is based, in part, on the development of a novelmethod for preparing crystals of ribosomes. The novel method providescrystals of the 50S ribosomal subunit that are much thicker than thoseavailable earlier and that can diffract X-rays to a resolution of about2.4 Å. The method eliminates the twinning of crystals that obstructedprogress in determining the crystal structure of the 50S ribosomalsubunit from H. marismortui for many years. The method of preparing thecrystals of the 50S ribosomal subunit is discussed below.

The present invention is also based, in part, on the atomic structure ofthe crystal of the 50S ribosomal subunit from H. marismortui that hasbeen derived from a 2.4 Å resolution electron density map that wasexperimentally phased using heavy atom derivatives. The atomicco-ordinates defining the large ribosomal unit were deposited on Jul.10, 2000, at Research Collaboratory for Structural Bioinformatics (RCSB)Protein Data Bank (PDB) (Berman et al. (2000) Azicleic Acid Research 28,235-242; http://www.rcsb.org/pdb/) with accession number PDB ID: 1FFK.

Moreover, the present invention is based, in part, on the derivationfrom the atomic co-ordinates of the following model which is brieflysummarized here and discussed in detail in the following sections of thespecification. This model includes 2,811 of the 2,923 nucleotides of 23SrRNA, all 122 nucleotides of its 5S rRNA, and structures for the 27proteins that are well-ordered in the subunit.

The secondary structures of both 5S and 23S rRNA are remarkably close tothose deduced for them by phylogenetic comparison. The secondarystructure of the 23S rRNA divides it into 6 large domains, each of whichhas a highly asymmetric tertiary structure. The irregularities of theirshapes notwithstanding, the domains fit together in an interlockingmanner to yield a compact mass of RNA that is almost isometric. Theproteins are dispersed throughout the structure, concentrated largely onits surface, but they are much less abundant in the regions of thesubunit that are of primary functional significance to proteinsyntheses—the 30S subunit interface, the binding regions for tRNA andthe peptidyl transferase active site. The most surprising feature ofmany of these proteins are the extended, irregular structures of theirloops and termini, which penetrate between RNA helices. The primary roleof most of the proteins in the subunit appears to be stabilization ofthe three-dimensional structure of its rRNA.

1. Preparation of the Crystal for the 50S Ribosonial Subunit andStructure Determination.

Several experimental approaches were used to extend the resolution ofthe electron density maps of the H. marismortui 50S ribosomal subunitfrom 5 Å to 2.4 Å including improvements in the crystals. Aback-extraction procedure was developed for reproducibly growingcrystals that are much thicker than those available earlier and candiffract to 2.2 Å resolution (see Example 1). Briefly, the crystals weregrown at room temperature in hanging drops by vapor diffusion fromseeded solutions back-extracted from precipitated subunits. The crystalsthat resulted had maximum dimensions of 0.5×0.5×0.2 mm and wereharvested after three weeks. The twinning of crystals that obstructedprogress for many years (Ban et al. (1999) supra) was eliminated byadjusting crystal stabilization conditions (see Example 1). Crystalswere stabilized by gradual transfer into a solution containing 12%, PEG6000, 22% ethylene glycol, 1.7 M NaCl, 0.5 M NH₄Cl, 100 mM potassiumacetate, 30 mM MgCl₂ and 1 mM CdCl₂, pH 6.2, and flash frozen in liquidpropane. Reducing the salt concentration below 1.7 M NaCl (KCl)increased the tendency of crystals to become twinned. At saltconcentrations as low as 1.2 M nearly all of the crystals were twinned.

All the X-ray data used for high resolution phasing were collected atthe Brookhaven National Synchrotron Light Source except for two nativedata sets used, which were collected at the Advanced Photon Source atArgonne (see Example 2) (Table 1). Osmium pentamine (132 sites) andIridium hexamine (84 sites) derivatives proved to be the most effectivein producing both isomorphous replacement and anomalous scattering phaseinformation to 3.2 Å resolution (see Example 2). Inter-crystal densityaveraging which had contributed significantly at lower resolution, wasnot helpful beyond about 5 Å resolution. Electron density maps weredramatically improved and their resolutions extended, eventually to 2.4Å, using the solvent flipping procedure in CNS (Abrahams et al. (1996)Acta Crystollogr. D 52: 30; Brünger et al. (1998) Acta Crystallogr. DBiol. Crystallogr. 54: 905-921). TABLE 1 Statistics for Data Collection,Phase Determination, and Model Construction Data Statistics MIRAS1MIRAS2 Native1 Os(NH₃)₅ ²⁺ UO₂F₅ ³⁻ Native2 Ir(NH₃)₆ ³⁺ Os(NH₃)₆ ³⁺Ta₆Br₁₂ ²⁺ Heavy atom — 30.0 0.5 — 20.0 4.5 3.0 conc. (mM) Soaking time(hrs) — 1.5 4 — 24 hrs 24 hrs 24 hrs Sites no. — 132 20 — 84 38 9Resolution (Å)  90-2.4  40-3.5  40-3.8  30-2.9  30-3.2  30-3.5  30-3.8(*) (2.5-2.4) (3.6-3.5) (3.9-3.8) (3.0-2.9) (3.27-3.20) (3.6-3.5)(3.97-3.80) (3.32-3.22) λ(Å) 1.00 1.14 1.30 1.00 1.075 1.14 1.255Observations 6,089,802 1,308,703 596,166 2,832,360 1,823,861 1,646,4681,288,524 Unique 665,928 429,761 313,863 390,770 541,488 488,275 346,745Redun. (*) 9.1 (6.5) 3.0 (2.5) 1.9 (1.6) 7.2 3.4 4.3 (4.2) 3.7Completeness (*) 95.6 (71.0) 99.4 (96.8) 92.0 (54.1) 97.1 93.8 98.1(99.0) 99.5 I/σI (Last bin) 25.5 (1.9)  13.5 (3.3)  8.9 (1.6) 18.0 (6.4)12.0 (2.6)  10.6 (2.7)  10.8 (3.2)  R_(merge) (*)  8.6 (69.1)  7.2(32.0)  9.1 (37.9)  11.2 (36.9)  8.5 (29.5) 12.1 (46.0) 12.1 (40.5) 02(ano) (*) — 2.8 (1.0) 1.5(1.0) — 2.63 (1.48) 1.8 (1.0) 2.42 (1.18)R_(merge) (ano) — 6.2 8.0 — 6.7 6.9 R_(iso) — 14.1 (22.7) 26.4 (47.0) —12.9 (28.1) 19.5 (39.4) Phasing Statistics Resolution shells (Å):˜73,200 reflections per bin 30.0 5.1 4.0 3.5 3.2 Total MIRAS1 (FOM) 0.520.31 0.14 — 0.32 Os(NH₃)₅ ²⁺ Phasing power 0.87 0.72 0.66 — 0.75 Phasingpower (SAD) 1.40 0.58 0.26 — 0.75 R_(cullis) (centric) 0.62 0.65 0.67 —0.65 UO₂F₅ ³⁻ Phasing Power 0.47 0.33 0.28 — 0.36 Phasing power (SAD)0.46 0.25 — — 0.66 R_(cullis) (centric) 0.72 0.77 0.75 — 0.75 MIRAS2(FOM) 0.48 0.40 0.28 0.12 0.33 Ir(NH₃)₆ ³⁺ Phasing power 1.02 0.92 0.780.66 0.89 Phasing power (SAD) 2.02 1.60 1.22 0.83 1.47 R_(cullis)(centric) 0.58 0.63 0.70 0.74 0.63 Os(NH₃)₆ ³⁺ Phasing power 0.62 0.570.58 0.58 0.59 Phasing power (SAD) 0.47 0.39 — — 0.42 R_(cullis)(centric) 0.78 0.78 0.78 0.76 0.78 Ta₆Br₁₂ ²⁺ (Used for SAD phasingonly) Phasing Power (SAD) 2.77 0.35 0.13 — 1.19 FOM (MIRAS1+HMIRAS2+SAD)0.76 0.51 0.31 0.14 0.37 Model Statistics Resolution range (Å)  90.0-2.4 rms deviations: Average B factors (Å²) Reflections 577,304Bonds (Å) 0.0064 All atoms 37.4 R_(sryst) (%) 25.2 Angles (°) 1.19 23SrRNA 32.3 R_(free) (%) 26.1 Dihedrals (°) 28.8 5S rRNA 43.2 Impropers(°) 1.68 Minimum/Max B factors (Å²) 70/107.9λ, wavelength; Redun., redundancy; (*) last-resolution shell.R_(iso): Σ|F_(PH) − F_(P)|/ΣF_(PH), where F_(PH) and F_(P) are thederivative and the native structure factor amplitudes, respectively.R_(sym): ΣΣ_(i)|I_((h)) − I_((h)i)|/ΣΣ:I_((h)i), where I(h) is the meanintensity after reflections. Phasing power: r.m.s. isomorphousdifference divided by the r.m.s. residual lack of closure.R_(cullis): Σ(∥F_(PH) − F_(P)| − |F_(H(calc))∥)/Σ|F_(PH) − F_(P)|, whereF_(PH) is the structure factor of the derivative and F_(P) is that ofthe native data. The summation is valid only for centric reflection.FOM (figure of merit): mean value of the cosine of the error in phaseangles.Abbreviations: MIRAS: multiple isomorphous replacement, anomalousscattering;SAD: single wavelength anomalous diffraction;FOM: figure of merit

Except for regions obscured by disorder, the experimentally-phased, 2.4Å resolution electron density map was of sufficient quality so that bothprotein and nucleic acid sequencing errors could be identified andcorrected. Each nucleotide could be fitted individually and thedifference between A and G was usually clear without reference to thechemical sequence, as was the distinction between purines andpyrimidines (FIG. 1).

Subtraction of the atomic model from the experimental electron densitymap leaves no significant density except for water and ions, showingthat the model accounts for all the macromolecular density. Preliminaryrefinement of the model was achieved using a mixed target in the programCNS (Brunger et al. (1998) supia). The model was further refined in realspace against the 2.4 Å electron density map using the program TNT(Tronrud (1997), Macromolecular Crystallography, Part B, Methods InEnzymology), which yielded a model with a free R-factor of 0.33. Oneadditional round of mixed target refinement of both atomic positions andB-factors using CNS led to the structure described below. Its freeR-factor is 0.27 (Table 1).

2. Sequence Fitting and Protein Identification.

The sequence of 23S rRNA was fit into the electron density mapnucleotide by nucleotide starting from its sarcin/ricin loop sequence(A2691-A2702) (E. coli numbers A2654 to A2665), whose position had beendetermined at 5 Å resolution (Ban et al. (1999) supra). Guided by theinformation available about the secondary structures of 23S rRNAs(Gutell, R. R. (1996), “Comparative Sequence Analysis and the Structureof 16S and 23S rRNA,” Ribosomal RNA. Structure, Evolution Processing,and Function in Protein Biosynthesis, (Dahlberg A. and Zimmerman B.,eds.), CRC Press, Boca Raton, Fla. pp. 111-128), the remaining RNAelectron density neatly accommodated the sequence of 5S rRNA. Theinterpretation of protein electron density corresponding to the proteinwas more complicated because each protein region had to be identifiedchemically before the appropriate sequence could be fit into it, butabout 4,000 amino acid residues were fit into electron density.

The H. marismortui 50S subunit appears to contain thirty-one proteins,and there are sequences in the Swiss-Prot data bank for twenty eight ofthose thirty one proteins, including one, HMS6 or L7ae, that wasoriginally assigned to the small ribosomal subunit (Whittmann-Liebold etal. (1990) supra). The three remaining proteins were identified usingthe sequences of the ribosomal proteins from eukaryotes and otherarcheal species as guides. No electron density was found for one of theH. marismortui large ribosomal subunit proteins in the sequencedatabase, LX. Either the assignment of LX to the large subunit is inerror, or LX is associated with a disordered region of the subunit, orLX is absent from the subunits examined altogether.

The 2.4 Å resolution electron density map lacks clear electron densityfor proteins L1, L10, L11 and L12, the positions of which are known fromearlier low resolution X-ray and/or electron microscopic studies. Theseproteins are components of the two lateral protuberances of the subunit,which are both poorly ordered in these crystals. L1 is the sole proteincomponent of one of them (Oakes, M. et al. (1986)), Structure Functionand Genetics of Ribosomes, (Hardesty, B. and Kramer, G., eds.)Springer-Verlag, New York, N.Y., 47-67) and is evident in 9 Å resolutiondensity maps of the subunit (Ban et al. (1998) supra), but not at higherresolutions. L10, L11 and L12 are components of the other protuberance,which is often referred to as the L7/L12 “stalk” (Oakes et al. (1986)supia). L11 and the RNA to which it binds were located in the 5 Åresolution electron density map of the H. marismortui large subunit (Banet al. (1999) supra) using the independently determined crystalstructures of that complex (Conn G L et al. (1999) Science 284:1171-1174; Wimberly et al. (1999) Cell 97: 491-502). A protein fragment(about 100 residues) that is associated with the RNA stalk that supportsthe L11 complex can be seen in the 2.4 Å resolution map. Based onlocation, it must be part of L10. There is no electron densitycorresponding to L12 seen at any resolution, but the L12 tetramer isknown to be attached to the ribosome through L10, and the L10/L12assembly is known to be flexible under some circumstances (Moller et al.(1986) Structure, Function and Genetics of Ribosomes, supra, pp.309-325), which may explain its invisibility here.

The structures of eubacterial homologues of proteins L2, L4, L6, L14,and L22 have previously been determined in whole or in part (see, Table2). L2, L6 and L14 were initially located in the 5 Å resolution map (Banet al (1999) supra). L4 and L22 have now been identified and positionedthe same way. Electron density corresponding to most of the remainingproteins was assigned by comparing chain lengths and sequence motifsdeduced from the electron density map with known sequence lengths,guided by the information available about relative protein positions(Walleczek et al. (1988) EMBO J. 7: 3571-3576) and protein interactionswith 23S rRNA and 5S rRNA (Ostergaard et al. (1998) J. Mol. Biol. 284:227-240). Each of the protein electron density regions so identified iswell accounted for by its amino acid sequence.

The most interesting of the proteins identified by sequence similaritywas L7ae, which first appeared to be L30e. The L30e identificationseemed plausible because the structure of yeast L30 superimposes neatlyon the electron density of L7ae, and the structure of the RNA to whichL7ae binds closely resembles that of the RNA to which yeast L30 binds(Mao, H. et al. (1999) Nat. Struct. Biol. 6: 1139-1147). Nevertheless,the sequence of HMS6, which by sequence similarity is a member of theL7ae protein family, fits the electron density better. Four of the otherproteins identified by sequence similarity, L24e, L37e, L37ae, and L44e,contain zinc finger motifs. The rat homologues of L37e and L37ae werepredicted to be zinc finger proteins on the basis of their sequences(Wool et al. (1995) supra), and this prediction helped identify theirhomologues in H. marismortui. TABLE 2 Large Subunit Proteins fromHaloarcula Marismortui Interactions⁵ Name¹ Hmlg² Lgth³ Conf⁴ 1 2 3 4 5 65S Proteins L1* 211 glb. + none L2† RL8 239 glb + ext + + + + (L37ae) L3RL3 337 glb + ext + + + + L14, L24e, (L13) L4† RL4 246 glb + ext + + Å(L18e), (L24), (L37e) L5 RL11 176 glb + + L18 L6 RL9 177 glb Å Å + (L13)L10* RP0 348 glb? + L12 L11* RL12 161 glb + none L12* RL1/2 115 glb L10L13 RL13a 145 glb + Å Å (L3), (L6) L14 RL23 132 glb + + + L3, L24e L15RL27a 164 glb + ext + + + (L18e), (L32e) L18 RL5 186 glb + ext Å + + L5,L21e L19 RL19 148 glb + ext + + + Å none L22 RL17 154 glb + ext +Å + + + + none L23 RL23a 84 glb Å + L29, (L39e) L24 RL26 119 glb + ext +(L4) L29 RL35 70 glb + L23 L30 RL7 154 glb + + none L18e RL18 115 glb +(L4), (L15) L21e RL21 95 glb + + Å L18 L24e RL24 66 glb Å + L3, L14 L31eRL31 91 glb + + + none L32e RL32 240 glb Å + (L15) L37e RL37 56 glb +ext + + + Å (L4) L39e RL39 49 ext + + (L23) L44e RL36a 92 glb + ext +Å + (L15e) L7ae R17a 110 glb Å L15e L10e RL10 163 glb + + Å none L15eRL15 184 glb + ext + Å Å Å + (L44e), L7ae L37ae RL37a 72 glb + ext + + +L2The top block of proteins include all those known to have eubacterialhomologues of the same name. The second block lists proteins found inthe H. marismortui large ribosomal subunit that have only eukaryotichomologues (Wittmann-Liebold et al. (1990) supra). Their names are allfollowed by the letter “e” to distinguish them from eubacterial proteinsthat would otherwise have the same name.# The third block are large subunit proteins for which no H. marismortuisequence yet exists. They are identified by sequence homology usingstandard L names.¹The structures of all or part of homologues of the following proteinswere previously determined: L1 (Nevskaya et al. (2000) Struct. Fold.Des. 8: 363), L2 (Nakagawa, A. et al. (1999) EMBO J. 18: 1459-1467), L4(Wahl et al. (2000) EMBO J. 19: 807-818), L6 (Golden et al. (1993) EMBOJ. 12: 4901-4908), L11 (Conn et al (1999) supra; Wimberly et al. (1999)supra; Markus et al. (1997) Nature Struct.# Biol. 4: 70-77), L12 (Leijonmarck, M. et al. (1980) Nature 286:824-827), L14 (Davies et al. (1996) Structure 4: 55-66), L22 (Unge etal. (1998) Structure 6: 1577-1586), L30 (Wilson et al. (1986) Proc. Nat.Acad. Sci. USA 83: 7251-7255). All other structures, except 10, havebeen newly determined in this study.²Rat homologue. Rat equivalents to H. marismortui proteins are from (Maoet al. (1999) supra).³Sequence chain length.⁴Conformation: glb = globular; ext = extension⁵The protein interactions with the 6 domains of 23S rRNA, 5S rRNA andother proteins are specified. (+) implies that the interaction issubstantial. (Å) implies a weak, tangential interaction. Protein namesare shown in parentheses implies that the interactions are weak;otherwise, the interaction is substantial.*All entries so designated describe proteins that are not fullyrepresented in the electron density maps described here. The summaryinformation provided is derived from literature sources and is includedhere for completeness only.†The structure available for this protein in isolation does not includethe extension(s) reported here.

3. General Appearance of the Subunit.

In its crown view (see FIG. 2), the large ribosomal subunit, which isabout 250 Å across, presents its surface that interacts with the smallsubunit to the viewer with the three projections that radiate from thatsurface pointed up. Although the protuberance that includes L1 is notvisible in the 2.4 Å resolution electron density map, the structure ofL1, which has been determined independently (Nikonov et al. (1996) EMBOJ. 15: 1350-1359), has been positioned approximately in lower resolutionmaps (Ban et al. (1998) supra) and is included here to orient thereader. It is evident that, except for its two lateral protuberances,the large ribosomal subunit is monolithic. There is no hint of adivision of its structure into topologically separate domains. Inaddition, partly because it lacks obvious domain substructure but alsobecause it is so large, it is impossible to comprehend looking at it asa whole. In order to convey to the reader a sense of how it is puttogether, the subunit must be dissected into its chemical components.

4. RNA Secondary Structure.

All the base pairs in H. marismortui 23S rRNA stabilized by at least twohydrogen bonds were identified using a computer program that searchedthe structure for hydrogen bond donors and acceptors separated by lessthan 3.2 Å. Bases linked by at least two such bonds were consideredpaired if the angle between their normals was less than 45°, and theangle between bonds and base normals was also less than 45°. Based onthe results of this analysis, a secondary structure diagram has beenprepared in the format standard for 23S/28S rRNAs (see FIG. 3). Thesecondary structure predicted for this molecule by phylogeneticcomparison was remarkably accurate, but it did not find all of thetertiary pairings and failed to identify interactions involvingconserved bases. In addition to base pairs of nearly every type, the RNAcontains numerous examples of well-known secondary structure motifs suchas base triplets, tetraloops, and cross-strand purine stacks, but nodramatically new secondary structure motifs have been identified so far.

The secondary structure of this 23 S rRNA consists of a central loopthat is closed by a terminal stem, from which 11 more or lesscomplicated stem/loops radiate. It is customary to describe the moleculeas consisting of 6 domains, and to number its helical stems sequentiallystarting from the 5′ end (see FIG. 4) (Leffers et al. (1987) supra). Thedivision of the molecule into domains shown in FIG. 4 deviates fromstandard practice with respect to helix 25, which is usually consideredto be part of domain I. It is placed in domain II because it interactsmore strongly with domain II than it does with the other elements ofdomain I.

There are five sequences longer than 10 nucleotides in 23 S rRNA whosestructures cannot be determined from the 2.4 Å resolution map due todisorder. Altogether they account for 207 out of the 232 nucleotidesmissing from the final model. The disordered regions are: (1) all ofhelix 1, (2) the distal end of helix 38, (3) helix 43/44 to whichribosomal protein L11 binds, (4) the loop end of stem/loop 69, and (5)helix 76/77/78, which is the RNA structure to which L1 binds. Forcompleteness, these regions are included in FIG. 3 (in gray) with theirsecondary structures determined phylogenetically.

5. Overall Architecture of rRNA.

The six domains of 23S rRNA and 5S rRNA all have complicated, convolutedshapes that nevertheless fit together to produce a compact, monolithicRNA mass (see FIGS. 4(A) and 4(B)). Thus despite the organization of itsRNAs at the secondary structure level, in three-dimensions, the largesubunit is a single, gigantic domain. In this respect, it is quitedifferent from the small subunit, which is a flatter object that is notat all monolithic. Even in low resolution electron micrographs the smallsubunit consists of three structural domains, each of which, it turnsout, contains one of the three secondary structure domain of its RNA(Noller et al. (1990) The Ribosome: Structure, Function and Evolution,supra, pp. 73-92). This qualitative difference between the two subunitsmay reflect a requirement for conformational flexibility that is greaterfor the small subunit.

Domain I, which looks like a mushroom (see FIG. 4(E)), lies in the backof the particle, behind and below the L1 region. The thin part of thedomain starts in the vicinity of domain VI, which is where its first andlast residues are located. Helices 1 and 25 span the particle in theback and then the domain expands into a larger, more globular structurebelow and behind the L1 region.

The largest of the six 23S rRNA domains, domain II, which accounts formost of the back of the particle, has three protrusions that reachtowards the subunit interface side of the particle (see FIG. 4(F)). Oneof them (helix 42-44) is the RNA portion of the L7/L12 stalk, which isknown to interact with elongation factors, is not well-ordered in thesecrystals. The second domain II protrusion is helix 38, which is thelongest, unbranched stem in the particle. It starts in the back of theparticle, bends by about 90 degrees and protrudes towards the smallsubunit between domains V and 58 rRNA. The third region, helix 32-35.1,points directly towards the small subunit and its terminus, the loop ofstem/loop 34, interacts directly with the small ribosomal subunit(Culver et al. (1999) Science 285: 2133-2135). This loop emerges at thesubunit interface between domains III and IV.

Domain III is a compact globular domain that occupies the bottom leftregion of the subunit in the crown view (see FIG. 4(G)). It looks like afour pointed star with the origin of the domain (stem/loop 48) andstem/loops 52, 57, and 58 forming the points. The most extensivecontacts of domain III are with domain II, but it also interacts withdomains I, IV and VI. Unlike all the other domains, domain III hardlyinteracts with domain V at all; the sole contact is a single van derWaals contact involving a single base from each domain.

Domain IV accounts for most of the interface surface of the 50S subunitthat contacts the 30S subunit (see FIG. 4(H)). It forms a large diagonalpatch of flat surface on that side of the subunit, and connects todomains III and V in the back of the particle. Helices 67-71 are themost prominent feature of domain IV, and form the front rim of theactive site cleft, which is clearly visible at low resolution (see FIG.2). This is one of the few regions of the 23S rRNA that is notextensively stabilized by ribosomal proteins. Helix 69 in the middle ofthis ridge interacts with the long penultimate stem of 16S rRNA in thesmall ribosomal subunit and can be viewed as a divider separating A-sitebound tRNAs from P-site bound tRNAs.

Domain V, which is sandwiched between domains IV and II in the middle ofthe subunit, is known to be intimately involved in the peptidyltransferase activity of the ribosome. Structurally the domain can bedivided into three regions (see FIGS. 4(I) and 4(J)). The first startswith helix 75 and ultimately forms the binding site for protein L1. Thesecond, which consists of helices 80-88, forms the bulk of the centralprotuberance region, and is supported in the back by the 5S rRNA anddomain II. The third region, which includes helices 89-93, extendstowards domain VI and helps stabilize the elongation factor bindingregion of the ribosome.

The smallest domain in 23S rRNA, domain VI, which forms a large part ofthe surface of the subunit immediately below the L7/L12 stalk, resemblesa letter X with a horizontal bar at the bottom (see FIG. 4(K)). Aninteresting region of this domain is the sarcin-ricin loop (SRL)(stem/loop 95), the structure of which has been extensively studied inisolation (Szewczak et al. (1995) J. Mol. Biol. 247: 81-98). The SRL isessential for factor binding, and ribosomes can be inactivated by thecleavage of single covalent bonds in this loop (Wool et al. (1992) TIBS17: 266-269). As suggested by nucleotide protection data, the majorgroove of this loop is exposed to solvent (Moazed et al. (1988) Nature334: 362-364), and its conformation is stabilized by proteins andthrough interaction with domain V that involves two bases on the minorgrove side. The nucleotides involved are A 2699 and G 2700 in domain VI,and A 2566 and G 2567 in domain V.

5S ribosomal RNA, which is effectively the seventh RNA domain in thesubunit, consists of three stems radiating out from a common junctioncalled loop A (see FIG. 4(D)). In contrast to what is seen in thecrystal structure of fragment 1 from E. coli 5S rRNA (Correll et al.(1997) Cell 91: 705-712), the helix 2/3 arm of the molecule stacks onits helix 4/5 arm, not helix 1 (see FIG. 4(L)). This arrangement resultsfrom a contorted conformation of loop A residues that involves twostacked base triples. Indeed, from the secondary structure point ofview, the loopA-helix 2,3 arm of 5S rRNA is quite remarkable. Nowhereelse in the subunit is there a higher concentration of unusual pairingsand of convoluted RNA secondary structure.

6. Sequence Conservation and Interactions in 23S rRNA.

While 23S/28S rRNAs contain many conserved sequences, they also varysubstantially in chain length. Shorter 23S/28S rRNAs are distinguishedfrom their longer homologues by the truncation of, or even theelimination of entire stem/loops, and by comparing sequences, one canidentify a minimal structure that is shared by all (Gerbi (1995)Ribosomal RNA: Structure, Evolution Processing and Function in ProteinBiosynthesis, supra, pp. 77-88). The expansion sequences in the 23S rRNAof H. marismortui, i.e., the sequences it contains that are larger thanthe minimum, are shown in FIG. 5 in green. They are largely absent fromthe subunit interface surface of the particle, but they are abundant onits back surface, far from its active sites. This is consistent with lowresolution electron microscopic observations suggesting that the regionof the large subunit whose structure is most conserved is the surfacethat interacts with the small subunit (Dube et al. (1998) Structure 6:389-399).

There are two classes of conserved sequences in 23S rRNA. One containsresidues concentrated in the active site regions of the large subunit.The second class consists of much shorter sequences scattered throughoutthe particle (FIG. 5: red sequences). The SRL sequence in domain VI andthe cluster of conserved residues belonging to domain V that are locatedat the bottom of the peptidyl transferase cleft are members of the firstclass. They are conserved because they are essential for substratebinding, factor binding and catalytic activity. Most of the residues inthe second class of conserved residues are involved in the inter- andintra-domain interactions that stabilize the tertiary structure of 23SrRNA. Adenosines are disproportionately represented in this class. Thepredominance of A's among the conserved residues in rRNAs has beenpointed out in the past (Ware et al. (1983) Nucl. Acids. Res. 22:7795-7817).

In addition to its reliance on A-dependent motifs, the tertiarystructure of the domains of 23S rRNA and their relative positions arestabilized by familiar tertiary structure elements like RNA zippers andtetraloop/tetraloop receptor motifs (Moore, P. B. (1999) Annu. Rev.Biochem. 68: 287-300), and in many places, base pairs and triplesstabilize the interactions of sequences belonging to differentcomponents of the secondary structure of 23S rRNA.

Interestingly, 5S rRNA and 23S rRNA do not interact extensively witheach other. The few RNA/RNA interactions there are involve the backbonesof the helix 4/5 arm of 5S rRNA and the backbone of helix 38 of 23SrRNA. Most of the free energy and all of the specificity of 5S rRNAbinding to the large ribosomal subunit appears to depend on itsextensive interactions with proteins that act as modeling clay stickingit to the rest of ribosome.

7. Proteins hi the 50S Ribosonial Subunit.

The structures of twenty seven proteins found in the large ribosomalsubunit of H. marismortui (Table 2) have been determined. Twenty-one ofthese protein structures have not been previously established for anyhomologues, and the structures of the six that do have homologues ofknown structure have been rebuilt into the electron density map usingtheir H. marismortui sequences. In addition, there are structuresavailable for homologues of H. marismortui L1, L11 and L12, which cannotbe visualized in the 2.4 Å resolution electron density map. Only thestructure of L10 is still unknown among the thirty one proteins known tobe present.

Not every one of these structures is complete. For example, an entiredomain of L5 is missing from the electron density, presumably because ofdisorder. L32e is also noteworthy in this regard. About twenty residuesfrom its N-terminus are not seen in the electron density map and theelectron density map suggests that its C-terminal residue is covalentlybound to the most N-terminal of its visible residues.

Of the thirty large subunit ribosomal proteins whose structures areknown, 17 are globular proteins, similar in character to thousands whosestructures are in the Protein Data Bank (Table 2). The remainingthirteen proteins either have globular bodies with extensions protrudingfrom them (“glb+ext”) or are entirely extended (“ext”). Their extensionsoften lack obvious tertiary structure and in many regions are devoid ofsecondary structure as well (see FIG. 6). These extensions may explainwhy many ribosomal proteins have resisted crystallization in isolation.The exceptions that prove the rule are L2 and L4, both of which areproteins belonging to the “glb+ext” class. Protein L2 was crystallizedand its structure solved only after its extensions had been removed(Nakagawa et al. (1999) supra), and one of the two regions of L4 thatare extended in the ribosome is disordered in the crystal structure ofintact L4 (Wahl et al. (2000) supra).

Except for proteins L1, L7, L10 and L11, which form the tips of the twolateral protuberances, the proteins of the 50S subunit do not extendsignificantly beyond the envelope defined by the RNA (see FIG. 7). Theirglobular domains are found largely on the particle's exterior, oftennestled in the gaps and crevices formed by the folding of the RNA. Thus,unlike the proteins in spherical viruses, the proteins of the largeribosomal subunit do not form a shell around the nucleic acid with whichthey associate, and unlike the proteins in nucleosomes, they do notbecome surrounded by nucleic acid either. Instead, the proteins act likemortar filling the gaps and cracks between “RNA bricks”.

The distribution of proteins on the subunit surface is nearly uniform,except for the active site cleft and the flat surface that interactswith the 30S subunit. In the crown view the proteins lie around at theperiphery of the subunit (see FIG. 7(A)), but when viewed from the sideopposite the 30S subunit binding site (the “back side”), they appear toform an almost uniform lattice over its entire surface (see FIG. 7(B)).Similarly, the bottom surface of the subunit, which includes the exit ofpolypeptide tunnel, is studded with proteins (see FIG. 7(C)). Indeed,the 5 proteins that surround the tunnel exit may play a role in proteinsecretion since they are part of the surface that faces the membrane andthe translocon when membrane and secreted proteins are beingsynthesized.

Although FIG. 7 shows protein chains disappearing into the ribosomeinterior, the degree to which proteins penetrate the body of theparticle can only be fully appreciated when the RNA is stripped away.The interior of the particle is not protein-free, but it is protein-poorcompared to the surface of the particle. Extended tentacles ofpolypeptide, many of which emanate from globular domains on the surface,penetrate into the interior, filling the gaps between neighboringelements of RNA secondary structure (see FIG. 8(E)). The bizarrestructures of these extensions are explained by their interactions withRNA.

Although extended, non-globular structures are rare in the protein database, they are not unknown. Extended protein termini often forminter-protein contacts, e.g., in viral capsids, presumably adoptingfixed structures only upon capsid formation. The basic “tails” ofhistones may behave the same way when nucleosomes form. The N-terminalsequences of capsid proteins are often positively charged, and in viruscrystal structures, the electron density for these sequences oftendisappears into the interior of the virus where these sequencespresumably interact with asymmetrically arranged nucleic acid. Theinteractions observed in the ribosome could be useful models for theseviral interactions.

The interactions of extended polypeptides and RNA in the large subunit,which stabilizes its massive nucleic acid structure, result in a tangleof RNA and protein in the center of the subunit (see FIGS. 8(A) and8(B)). It is hard to imagine such an object assembling from itscomponents efficiently in anything other than a highly ordered manner.Chaperones may well be required to prevent the aggregation of theextended regions of these proteins, which are likely to be disorderedoutside the context provided by rRNA, and to manage the folding of rRNA.

8. Protein and RNA Interactions.

Because protein permeates the large subunit to a surprising degree,there are only a few segments of the 23S rRNA that do not interact withprotein at all. Of the 2923 nucleotides in 23S rRNA, 1157 nucleotidesmake at least van der Waals contact with protein (see FIG. 8(D)), andthere are only ten sequences longer than twenty nucleotides in which nonucleotide contacts protein. The longest such sequence containsforty-seven nucleotides, and is the part of domain IV that forms theridge of the active site cleft.

The extent of the interactions between RNA and protein that occur whenthe large subunit assembles can estimated quantitatively. Using theRichards algorithm (Lee, B. et al. (1971) J. Mol. Biol. 55: 379-400) anda 1.7 Å radius probe to compute accessible surface areas, it can beshown that 180,000 Å² of surface becomes buried when the subunit formsfrom its isolated, but fully structured components. This is about halftheir total surface area. The average is about 6,000 Å² per protein.While this is an enormous amount compared to the surface buried whenmost protein oligomers form, it should be recognized that ribosomeassembly must be accompanied by a large loss in conformational entropythat does not occur when most proteins oligomerize. The extended proteintermini and loops of the ribosomal proteins are almost certainlyflexible in isolation, and in the absence of protein, the RNA isprobably quite flexible as well. Thus, the burial of a large amount ofsurface area may be required to provide the energy necessary to fix ofthe structures of these molecules.

All of the proteins in the particle except L12, interact directly withRNA and all but seven of the remaining thirty proteins interact with tworRNA domains or more (Table 2). The “champion” in this regard is L22,which is the only protein that interacts with RNA sequences belonging toall 6 domains of the 23S rRNA (see FIG. 8(C)). The protein-mediatedinteractions between 5S rRNA and 23S rRNA are particularly extensive.Protein L18 attaches helix 1 and helix ⅔ of 5S rRNA to helix 87 of 23SrRN-A. Protein L31e mediates an interaction between the same part of 5SrRNA and domains II and V. Loop C is linked to domain V by protein L5and loop D is attached to domains II and V by protein L10e. Whateverelse they may do, it is evident that an important function of theseproteins is stabilization of the relative orientations of adjacent RNAdomains. Several also help secure the tertiary structures of the domainswith which they interact.

Since most ribosomal proteins interact with many RNA sequences and thenumber of proteins greatly exceeds the number of RNA domains, it canhardly come as a surprise that every rRNA domain interacts with multipleproteins (Table 2). Domain V, for example, interacts with thirteenproteins, some intimately and a few in passing.

It is clear that the oligonucleotide binding experiments long relied onfor information about the RNA binding properties of ribosomal proteinshave underestimated their potential for interacting with RNA. Thehigh-affinity RNA binding site identified on a protein by such anexperiment may indeed be important for ribosome assembly, but its many,weaker interactions with other sequences are likely to be missed, andthey too may be vital for ribosome structure. Most ribosomal proteinscrosslink RNA and crosslinking is impossible without multipleinteractions. Similar considerations may apply to proteins that arecomponents of other ribonucleoproteins such as the sliceosome.

Of the seven proteins that interact with only one domain, three (L1,L10, L11) participate directly in the protein synthesis process. Ratherthan being included in the ribosome to ensure that the RNA adopts theproper conformation, it seems more appropriate to view the RNA as beingstructured to ensure their correct placement. Another three (L24, L29,L18e) interact with several secondary structure elements within thedomains to which they bind, and presumably function to stabilize thetertiary structures of their domains. The last of the single RNA domainproteins, L7ae, is puzzling. On the one hand, it cannot function as anRNA stabilizing protein because it interacts with only a single, shortsequence in domain I, and on the other hand, it is far from theimportant functional sites in the subunit, the peptidyl transferase siteand factor binding site. It is quite close to L1, however, which appearsto be important for E-site function (Agrawal et al (1999) J. Biol. Chem.274: 8723-8729), and maybe it is involved in that activity.

While many ribosomal proteins interact primarily with RNA, a fewinteract significantly with other proteins. The most striking structuregenerated by protein-protein interactions is the protein clustercomposed of L3, L6, L 14, L19 and L24e that is found close to the factorbinding site. The protein surface they provide may be important forfactor interactions.

The structure presented above illuminates both the strengths andweaknesses of approaches to complex assemblies that depend ondetermining the structures of components in isolation. The structures ofthe globular domains of homologues of the proteins in large ribosomalsubunit from H. marismortui are largely the same as those of thecorresponding domains in the intact subunit, though adjustments indomain positions are sometimes required. Consequently, these structureswere very useful for locating proteins and interpreting lower resolutionelectron density maps. However, for obvious reasons, the structures ofthe extended tails and loops of ribosomal proteins cannot be determinedin the absence of the RNAs that give them structure, and the feasibilityof strategies that depend on producing low molecular weight RNA-proteincomplexes that have all the RNA contacts required to fix the structuresof such proteins seems remote. RNA is also a problem. While thesarcin/ricin loop has much the same structure in isolation as it does inthe ribosome, the structure of 5S rRNA in isolation differs in somerespects from what is seen in the ribosome, and the structure of theisolated P-loop (Puglisi et al. (1997) Nat. Struct. Biol. 4: 775-778)does not resemble the structure of the P-loop in the ribosome at all.Clearly a “structural genomics” approach to the ribosome, which wouldhave entailed determining the structures of all its proteins and allpossible rRNA fragments, would not have worked. It may not be successfulfor other macromolecular assemblies either.

B. The Structural Basis of Ribosome Activity in Peptide Bond Synthesis

Analysis of the atomic co-ordinates discussed in section IIA abovetogether with additional atomic co-ordinates of a ribosomal subunitcomplexed with various analogues, similarly refined, permit an analysisof ribosome function. Accordingly, the present invention is also basedon the crystals of Haloarcula marismortui 50S ribosomal subunitcomplexed either with the Yarus transition state analogue, CCdA-p-Puro,or with a mini-helix analogue of an aminoacyl-tRNA. The presentinvention provides the structures of both complexes. The atomicco-ordinates of the structure of both complexes were deposited on Jul.26, 2000, at Research Collaboratory for Structural Bioinformatics (RCSB)Protein Data Bank (PDB) (Berman et al (2000) Nucleic Acid Research 28:235-242; http://www.rcsb.org/pdb/) with accession numbers PDB ID: 1FFZ(50S ribosome/CCdA-p-Puro complex) and PDB ID: 1FG0 (50Sribosome/aa-tRNA analogue).

As discussed below, the complete atomic structures of the largeribosomal subunit and its complexes with two substrate analogues showthat the ribosome is a ribozyme. The complete atomic structures alsoprovide information regarding the catalytic properties of its all-RNAactive site. Both substrate analogues are contacted exclusively byconserved rRNA residues from domain V of 23S rRNA; there are no proteinside-chains closer than about 18 Å to the peptide bond beingsynthesized. The mechanism of peptide bond synthesis appears to resemblethe reverse of the deacylation step in serine proteases, with the baseof A2486 (A2451) in E. coli playing the same general base role as His57in chymotrypsin. The unusual pKa that A2486 must possess to perform thisfunction probably derives from its hydrogen bonding to G2482 (G2447)which interacts with a buried phosphate that could stabilize the unusualtautomers of two bases which is required for catalysis. The polypeptideexit tunnel is largely formed by RNA but has significant contributionsfrom proteins L22, L39 and L4 and its exit is encircled by proteins L19,L22, L23a, L24 and L29.

The CCdA from the Yarus analogue binds to the so-called P-loop and hencemust be in the P-site. Only the terminal-CCA of the aa-tRNA analogue isvisible, but since it interacts appropriately with the A-loop (Kim etal. (1999) Molec. Cell 4: 859-864), it must be in the A-site. Thepuromycin group occupies the same location in both structures, and thereare no proteins near that site. Hence, the catalytic activity of theactive site must depend entirely on RNA. The N3 of A2486 (E. coli A2451)is the titratable group nearest to the peptide bond being synthesizedand is likely functioning as a general base to facilitate thenucleophilic attack by the α-amino group of the A-site substrate. Inorder to function in this capacity, the pKa of this base has to beroughly 5 units higher than normal.

1. Structures of Substrate Analogue Complexes.

In order to establish how substrates interact at the A-site and P-siteof the large subunit, two substrate analogues were used. One of theanalogues, which was designed to mimic the acceptor stem of an aa-tRNAand bind to the A-site, was a twelve base-pair RNA hairpin with anaminoacylated, four-nucleotide extension on its 3′ end (see FIG. 9). Thesequence used was that of the tRNA tyr acceptor stem and it isterminated with puromycin, which itself is an analogue of tyrosyl-A76.The second analogue used was the Yarus transition state analogue,CCdA-p-puromycin. As in the case of the A-site substrate analogue, thepuromycin of the Yarus inhibitor is expected to bind at the A-site,while its CCdA moiety should bind at the P-site.

The positions of the Yarus inhibitor and the tRNA acceptor stem analoguewere determined by soaking these molecules into crystals of the H.marismortui 50S ribosomal subunit, measuring diffraction data to 3.2 Åresolution and calculating difference electron density maps (Welch etal. (1997) Biochemistry 36: 6614-6623). Maps of the complexes were alsocalculated using 2F_(o)(complexed)-F_(o)(uncomplexed) as coefficients,to examine the shifts in the positions of ribosome residues that occurwhen these analogues bind (see FIG. 10(B) and Table 3). TABLE 3Statistics for Data Collection and Scaling. Crystal Native A Native BCcdAp-Puro Mini-helix Soak time (hours) — — 24 24 Soak concentration(μM) — — 100 100 Wavelength (Å) 1.0 1.0 1.0 1.0 Observations 1,571,1711,344,877 2,590,726 2,712,813 Unique 284,533 369,167 367,284 447,121Redundancy 5.5 3.6 7.0 6.0 Resolution limits (Å) 70.0-3.2  70.0-3.0 70.0-3.0  70.0-2.8  (High-resolution bin)* (3.26-3.20) (3.05-3.00)(3.23-3.17) (3.08-3.02) Completeness 94.1 (96.0) 98.9 (99.3) 98.6 (99.9)99.6 (100)  l/σl 14.6 (4.0)  10.8 (3.1)  11.0 (2.8)  10.7 (2.9) R_(merge) † 10.2 (40)   11.5 (38)   18.8 (84)   14.3 (72)   R_(iso)Native A‡ — —  6.8 (20.8) 14.4 (25.2) R_(iso) Native B‡ — — 12.6 (27.4)17.5 (31.0)*Statistics in parenthesis are calculated for the high-resolution binused in map calculations, which, as indicated was sometimes lower inresolution than the high-resolution bin used in data reduction.†R_(merge): ΣΣ_(i)|I_((h)) − I_((h)i)|IΣΣI_((h)i)′ where I_((h)) is themean intensity after reflection.‡R_(iso): Σ|F_(PH) − F_(P)|ΣF_(PH)_where F_(PH) and F_(P) are the soakedand the native crystal structure factor amplitudes respectively.

A model for the entire Yarus inhibitor could be fitted into thedifference density (see FIG. 10(A)), and the electron density map of thecomplex shows the N3 of A2486 (2451) within hydrogen bonding distance ofa non-bridging oxygen of the phosphoramide (see FIG. 10(B)). Theinhibitor's two C's, which correspond to C74 and C75 of peptidyl-tRNA,are Watson-Crick base-paired with G2285 (2252) and G2284 (2251) in theP-loop, respectively (see FIG. 11(A)). The C74-G2285 (2252) interactionwas predicted by the results of Noller and coworkers (Noller et al.(1992) Science 256: 1416-1419). The dA, which corresponds to A76 of atRNA in the P-site, is not base-paired, but rather stacks on the riboseof A2486 and hydrogen bonds to the 2′OH of nucleotide A2485 (see FIG.12(B)).

Only the CC-puromycin moiety of the mini-helix acceptor stem analogueshowed ordered electron density in its difference electron density map(see FIG. 10(C)). The C75 of the acceptor stem CCA is Watson-Crickbase-paired with G2588 (2553) of the A-loop, whereas the C74 is moredisordered and not base-paired but appears to stack on a ribosome base.The dimethyl A of the A-site inhibitor puromycin is positionedidentically to the dimethyl A of the Yarus inhibitor. Further, thedimethyl A of puromycin, which is the A76 equivalent of an A-site tRNA,interacts with the A-loop in much the same way that the A76 from theP-site CCA interacts with the P-loop (see FIG. 11(B)).

The most notable of the several conformational changes in the ribosomeinduced by the binding of the transition state analogue is the orderingof base A2637 (2602), which is disordered in the unliganded enzyme (seeFIG. 11(B)). It becomes positioned between the CCA bound at the A-siteand the CCA bound at the P-site. The base of U2620 (2585) also moves sothat it can make a hydrogen bond with the 2′ hydroxyl of the ribose ofA76 in the A-site, and U2619 and G2618 shift to allow the A76 to bepositioned. Smaller shifts in positions are observed in the positions ofA2486, whose N3 is near to the non-bridging oxygen of the phosphate, andone of the G residues with which it interacts, G2102 (2482).

2. Location and Chemical Composition of the Peptidyl Transferase Site.

The inhibitors are bound to a site made entirely of 23S rRNA with noproteins nearby, proving that the ribosome is a ribozyme. Both the Yarusinhibitor and the A-site analogue of aa-tRNA bind to the large subunitat the bottom of a large and deep cleft at the entrance to the 100 Ålong polypeptide exit tunnel that runs through to the back of thesubunit (see FIG. 12). This site is surrounded by nucleotides belongingto the central loop of 23S RNA domain V, the “peptidyl transferaseloop.” Nucleotides from the single stranded portions of this loop makethe closest approach to the phosphate that mimics the tetrahedral carbonintermediate. In general, the helices that extend from the peptidyltransferase loop in 2 secondary structure diagrams of 23S rRNA alsoextend away from the active site in the tertiary structure (see FIG.13). Although there are 13 proteins that interact with domain V (seeFIG. 14(A)), there are no globular proteins in the vicinity of theinhibitor. The closest polypeptides are the non-globular extensions ofseveral proteins (L2, L3, L4, L10e) that penetrate deeply into domain Vand approach the active site (see FIG. 14(B)). These extensions fillmany of the voids between the RNA helices of domain V, neutralizephosphate backbone charge, and presumably stabilize the structure of thedomain and its association with other RNA regions. However, none oftheir side chain atoms is closer than about 18 Å to the phosphorus ofthe inhibitor's phosphate group, which marks the site where peptidebonds form. Furthermore, the substrate analogue is completely enclosedin an rRNA cavity that is so tightly packed that there is no possibilitythat an unidentified peptide could be lurking nearby (see FIG. 15).Thus, the catalytic entity in the ribosome must be RNA.

Two of the proteins with long termini or loops penetrating the rRNAscaffold of domain V are proteins that could not previously be excludedfrom involvement in the peptidyl transferase reaction L2 and L3 (Noller(1991) Ann. Rev. Biochem. 60: 191-227). Noller and colleagues (Noller etal. (1992) supra) found that under conditions which prevent RNAdenaturation, extensive digestion of Thermus thermophilus 50S subunitswith proteases followed by extraction with phenol and other agents thatdisrupt protein-RNA interactions did not remove several peptides fromthe subunit that were less than 10,000 in molecular weight. Thestructure makes it clear why these protein fragments were particularlyresistant to protease treatments. While protease treatment could digestthe globular protein domains on the surface of the large subunit, itcould not remove the long termini or loops that penetrate deeply in the23S rRNA because they are sequestered within the rRNA and thus protectedfrom cleavage, independently of the globular domains.

3. Peptidyl Transferase Active Site.

The RNA that surrounds the substrate analogues is closely packed, muchlike the active site region of a protein enzyme and the nucleotides incontact with the inhibitor are greater than 95% conserved in all threekingdoms of life (see FIG. 15). Thus, it is clear that the ribosome is aribozyme, but what gives the RNA its catalytic power?

Without wishing to be bound by theory, the residue most likely to beinvolved in catalysis, presumably as a general base, is A2486, whose N3is about 3 Å from the phosphoramide oxygen of the Yarus inhibitor thatis the analogue of the carbonyl oxygen of a nascent peptide bond andabout 4 Å from the amide that corresponds to the amide nitrogen of thepeptide bond being synthesized. Ordinarily, the pKa of the N1 ofadenosine monophosphate is about 3.5 and that of its N3 is perhaps 2 pHunits lower (Saenger (1984) Principles of Nucleic Acid Structure, (C. R.Cantor, eds.), Springer Advanced Texts in Chemistry, Springer-Verlage,New York, N.Y.), and in order for A2486 to function as a general base,its pKa would have to be raised to 7 or higher. The crystal structureitself suggests that its pKa is, in fact, quite unusual. The N3 of A2486can only hydrogen bond to the phosphate oxygen, as observed, if it (orthe phosphate oxygen) is protonated. The distance between these twoatoms is about 3 Å indicating that a hydrogen bond does, indeed, existbetween them. Since the crystal is at pH 5.8, this implies that the pKaof the N3 is greater than 6. Muth and Strobel have measured the pKa ofthe corresponding A in E. coli 23S RNA by examining its dimethyl sulfatereactivity as a function of pH and have concluded that it is 7.6, thoughthey cannot be sure from their experiments whether it is the N3 or N1whose pKa they have measured (Muth et al. (2000) Science 289: 947-950).Because there is no other available, titratable RNA functional groupcloser than about 7 Å to the nascent peptide bond, there is not othergroup available to function as a general base.

There are several features of environment of A2486 (2451) that mightaffect its pKa. The pKa of the N3 of A2486 (2451) may be increasedsignificantly in part by a charge relay mechanism, analogous to thatwhich occurs in the active site of the serine proteases, with the buriedphosphate of A2485 (2450) performing a similar function as the buriedcarboxylate of Asp102 of chymotrypsin. The experimental 2.4 Å electrondensity map unambiguously establishes the hydrogen bonding interactionsin this most critical region of the active site (see FIG. 16(A)). The N6of A2486 interacts with the 06 atoms of G2482 (2447) and G2102 (2061)(see FIG. 16(B)). The N2 of G2482 is also interacting with anon-bridging oxygen of the phosphate group of A2485 (2450) that is amongthe total of 3 most solvent inaccessible phosphate groups (826, 1497 and2485) in the large ribosomal subunit for which we do not see anyneutralizing counterion in the 2.4 Å resolution map. Weak density thatmay correspond to a water molecule is hydrogen bonded to the othernon-bridging oxygen. A neutralizing counterion is not apparent in thisstructure. The buried phosphate of A2485 could abstract the proton fromthe exocyclic N2 of G2482 in order to neutralize its energeticallyunfavorable buried negative charge. This, in turn, would stabilize theotherwise rare imino tautomer of that base. The interaction of an iminoof G2482 with A2486 likewise can stabilize the imino tautomer of A2486that would result in a negative charge on its N3 were it unprotonated(see FIG. 16(C)). In this way, some of the negative electrostatic chargeoriginating on the buried phosphate of A2485 could be relayed to the N3of A2486, thereby increasing its pKa.

A second feature of the environment of the catalytic site that mayaffect its stability, tautomeric state and electrostatic chargedistribution is a bound monovalent cation. A potassium or a sodium ioninteracts with the 06 of G2482 and G2102 as well as with three otherbases. Its identity as a potassium ion is established by its observedcontinuation and by an independent experiment showing that a rubidiumion can bind to this site. The monovalent ion might also stabilizenon-standard tautomers, but its expected influence on the pKa of A2486is less obvious. Early biochemical experiments have shown the importanceof potassium for peptidyl transferase activity (Monro (1967) supra;Maden et al. (1968) supra) and this binding site could be responsiblefor that affect.

It may also be the case that stabilization of an imino tautomer by aburied phosphate explains the expected higher pKa of a catalyticcytosine in the active site of the hepatitis delta virus ribozyme(Ferre-D'Amare et al. (1998) Nature 395: 567-574; Naharo et al. (2000)Science 287: 1493-1497). In this case, a backbone phosphate, whosesolvent accessibility is similar to that of A2485 in the ribosome, isobserved to hydrogen bond to the N4 of C, and the protonated form of theimino tautomer of that C would neutralize the phosphate, promoting thefunction of its N3 as a general acid (Naharo et al. (2000) supra).

4. Catalytic Mechanism of Peptide Bond Formation.

The proximity of the N3 of A2486 (2451) to the peptide bond beingsynthesized and the nature of the reaction catalyzed suggest a chemicalmechanism of peptide synthesis that is analogous to the reverse of thedeacylation step seen in serine proteases during peptide hydrolysis(Blow et al. (1969) Nature 221: 337-340; Steitz et al. (1982) Ann. Rev.Biophys. Bioeng. 11: 419-444). In that mechanism, the basic form ofHis57 abstracts a proton from the α-amino group of the peptide productas it attacks the acyl-Ser195. Formation of the tetrahedral carbonylcarbon intermediate is stabilized by interaction of the oxyanion formedwith backbone amides (the “oxyanion hole”); His57 shuttles the protonacquired from the α-NH₂ to Ser195 as the tetrahedral intermediate breaksdown.

The residue A2486 (2451) appears to be the analogous to His57 inchymotrypsin and that the peptidyl-tRNA is analogous to acyl-Ser195.Thus, the N3 of A2486, with its greatly elevated pKa, abstracts a protonfrom the α-amino group of the A-site bound aminoacyl-tRNA facilitatingthe nucleophilic attack of that amino group on the carbonyl carbon thatacylates the 3′ OH of the tRNA in the P-site (see FIG. 17(A)). Incontrast to the serine proteases, however, the oxyanion of thetetrahedral intermediate is near to the protonated N3 of A2486 (A2451)rather than being proximal to a separate oxyanion binding site. Thus, itcould be that the protonated N3 of A2486 stabilizes the formation of theoxyanion by hydrogen bonding to it, as we observe in the Yarus inhibitorcomplex (see FIG. 17(B)). The N3 of A2486 could then subsequentlytransfer its proton to the 3′ hydroxyl of the P-site bound tRNA, whichis liberated as the peptide shifts to the A-site bound tRNA (see FIG.17(C)).

An additional question is how is the catalyzed hydrolysis of thepeptidyl tRNA in the P-site prevented prior to the delivery of the nextappropriate aa-tRNA to the A-site? It appears from this complex thatwater would not be excluded from access to the peptidyl link to theP-site tRNA if the A-site were vacant. An analogous problem wasdiscussed by Koshland in the 1960s (Koshland, Jr. (1963) Cold SpringHarbor Symp. Quant. Biol. 28: 473-489), who theorized why hexokinasedoes not hydrolyze ATP in the absence of glucose, since water shouldbind perfectly well to the binding site used by the 6-hydroxyl ofglucose. The answer proposed was induced fit, i.e., hexokinase is notcatalytically competent until the glucose binds and produces aconformational change that orients substrates and catalytic groupsoptimally. This indeed appears to be the case (Bennett, Jr. et al.(1978) Proc. Natl. Acad. Sci. USA 75: 4848-4852). Similarly, it could bethat the catalytic A2486 and/or the peptidyl substrate are not properlyoriented or that the binding site for the α-NH₂ group is blocked by areoriented ribosome base in the absence of aa-tRNA in the A-site. We doobserve that the base of U2620 is close to A2486 in the ligand freestructure, and it may serve as the appropriate plug that preventsspontaneous hydrolysis of peptidyl-tRNA.

Thus, it appears that this RNA enzyme uses the same principles ofcatalysis as a protein enzyme. First, a large catalytic enhancement isachieved by precisely orienting the two reactants, the αNH₂ from theA-site aminoacyl-tRNA and the carbonyl carbon from the P-sitepeptidyl-tRNA. This may be accomplished, in part, by the interactions ofthe CCA ends of the A-site and P-site tRNAs with the A-loop and P-loop,respectively. Secondly, acid-base catalysis and transition statestabilization are achieved by an enzyme functional group (A2486 (2451)in this case) whose chemical properties are altered appropriately by theactive site environment. Third, similar chemical principles may be usedby RNA and protein enzymes to alter the pKa's of functional groups. Aburied carboxylate of Asp102 acting through His57 alters thenucleophilicity of Ser195 in chymotrypsin (Blow et al. (1969) supra). Inthe ribosome a solvent inaccessible phosphate may act through G2482(2447) alters the nucleophilicity of the N3 of A2486 (2451). It could bethat RNA molecules “learned” how to use the chemical principles ofcatalysis significantly before protein molecules did.

5. tRNA Binding.

While it is not possible to bind tRNA molecules to either the A- orP-sites in these crystals for steric reasons, it is possible to placethe A-, P- and E-site tRNA molecules on the large ribosomal subunit inthe same relative orientation that Cate et al. observed in theircrystallographic study of the Thermus aquaticus 70S ribosome. Theco-ordinates of the three tRNA molecules in the relative positions seenin the 70S ribosome can be docked on the Haloarcula marismortui largeribosomal subunit in a way that avoids steric clashes and places theacceptor stems of the A-site and P-site tRNAs near to the positions ofthe CCAs we have observed bound to the A-loop and P-loop (see FIG. 18).Although nucleotides C74 and C75 were modeled in a differentconformation in the 7.8 Å ribosome map, the C74 residues from the CCAsin both the A- and P-sites can be connected to residue 72 of the dockedA-site and P-site tRNAs through a modeled residue 73, and it appearsthat the tRNA molecules fit well onto the surface of the subunit.Unexpectedly, this modeling places the E-site, P-site and A-site boundtRNA molecules in close proximity to three ribosonal proteins. ProteinsL5 and L10e are near tRNAs in the P-site and A-site. Since both of theseproteins also interact with 5S rRNA, this observation raises thepossibility that 5S rRNA and some of its associated proteins might helpstabilize the positioning of ribosome bound tRNAs and is consistent withthe fact that 5S rRNA enhances ribosomal activity, but is not absolutelyessential for it (Moore, Ribosomal RNA & Structure, Evolution,Processing and Function in Protein Biosynthesis (1996), supra, pp.199-236). Protein L44e appears to interact with the E-site tRNA and maycontribute to E-site activity. According to this docking experiment theA-site tRNA interacts with the highly conserved stem-loop 2502-2518(2467-2483) which together with L10e forms a large concave surface thatcontacts the tRNA on the T-stem, utilizing the exact same binding siteexploited by EF-Tu (Gutell et al. (2000) supra).

Examination of the relationships between the CCAs bound in the A- andP-sites and the tRNAs to which they are connected as well as theirinteractions with the ribosome also leads to some insights intotranslocation. Immediately after formation of the new peptide bond anddeacylation of the P-site tRNA, the acceptor end of the P-site tRNA isknown to move to the E-side and that of the A-site tRNA moves to theP-site (Blobel et al. (1970) J. Cell. Biol. 45: 130-145). Theapproximate modeling of the 3 tRNA molecules on the large subunitsuggests some possible contributions to this process. First, there aretwo base-pairs between the P-site tRNA and the P-loop and only onebetween the A-site and the A-loop. Moving from the A- to the P-siteincreases base-pairing, though there must be a concomitant attraction ofthe deacylated P-site tRNA to an E-site. Further, the CCAs bound to theA and P loops are related by 180° rotation, whereas the tRNAs to whichthey are attached are not. Thus, the relationships of the CCAs to theacceptor steins cannot be the same in both sites and may not be equallystable. If the conformation of the A-site tRNA is less stable, thenmoving a tRNA from the A- to the P-site might be energetically favored.

6. Polypeptide Exit Tunnel

It appears very likely from the structure that all nascent polypeptidespass through the polypeptide exit tunnel before emerging from theribosome, because there appears to be no other way out. We are now ableto address two important questions about the functioning of thepolypeptide exit tunnel: (1) Why do nascent proteins not stick to itswalls? Teflon has the marvelous property of not sticking to denaturedegg proteins, so how has the ribosome achieved a similar non-sticksurface for the denatured proteins that must pass through the tunnel?(2) Do proteins fold to any degree in the tunnel giving the ribosome achaperon-like function?

The length of the tunnel from the site of peptide synthesis to its exitis about 100 Å, broadly consistent with the length of nascentpolypeptide that is protected from proteolytic cleavage by the ribosome(Moazed et al. (1989) Nature 342:142) and the minimum length requiredfor antibody recognition at the exit (Picking et al. (1992) Biochemistry31: 2368-2375). The tunnel is largely straight, except for a bend 20 to35 Å from the peptidyl transferase center (see FIG. 19). Its diametervaries from about 20 Å at its widest to a narrow point of about 10 Å atthe very beginning and at a position 28 Å from the tunnel exit with anaverage diameter of about 15 Å. Since the smallest orifice through whichthe polypeptide product must pass only barely accommodates the diameterof an α-helix diameter, it seems unlikely that significant proteinfolding beyond the formation of α-helix could occur within the ribosome.

The majority of the tunnel surface is formed by domains I-V of 23S rRNA,but significant contributions are also made by the non-globular regionsof proteins L22, L4 and L39 which not only fill some of the voids in theRNA scaffold, but also form significant portions of the tunnel wall (seeFIG. 19). The largest protein contributor to the surface of the tunnelis L22 whose long α-hairpin loop lies between RNA segments of domains Ithrough IV and is approximately parallel with the axis of the tunnel.Unlike the other tunnel proteins, protein L39 does not have a globulardomain at the surface of the particle and is almost entirely buried indomains I and III underneath protein L23. Interestingly, the nucleotidesof 23S rRNA that form the tunnel wall are predominantly from loops inthe 23S rRNA secondary structure (see FIG. 19). As it progresses throughthe tunnel from the active site, a nascent polypeptide first encountersdomain V followed 20 Å further along by domains II and IV and proteinsL4 and L22. The last half of the tunnel is formed by domains I and IIIand the protein L39e.

The narrowest part of the tunnel is formed by proteins L22 and L4 whichapproach the tunnel from opposite sides forming what appears to be agated opening (see FIG. 19C). The function of this constriction, if any,is not obvious. It might be the place where the nature of the nascentchain is sensed and the information transmitted to the surface of theparticle, perhaps through L22 or L4. The α-hairpin of L22 at the site ofthis orifice and the 23S rRNA interacting with it are highly conserved;its globular portion is located adjacent to the tunnel exit on thesurface that must face the translocon during protein secretion (see FIG.19).

The “non-stick” character of the tunnel wall must reflect a lack ofstructural and polarity complementarity to any protein sequence orconformation that it encounters. The tunnel surface is largelyhydrophilic and includes exposed hydrogen bonding groups from bases,backbone phosphates and polar protein side-chains (see FIG. 19). Whilethere are many hydrophobic groups (sugars, bases, protein side-chains)facing the tunnel as well, there are no patches of hydrophobic surfacelarge enough to form a significant binding site for hydrophobicsequences in the nascent polypeptide. As the tunnel is some 20 Å indiameter and filled with water and the newly synthesized polypeptide ispresumably freely mobile, the binding of a peptide to the tunnel wallwould result in a large loss of entropy that would have to becompensated for by a large complementary interaction surface that islarger than 700 Å (Chothia et al. (1975) Nature 256: 705-708).Similarly, while Arg and Lys side-chains from a nascent peptide mayindeed interact with the phosphates exposed in the tunnel, the degree ofstructural complementarity and the net binding energy obtained afterdisplacing bound counterions must be too small to overcome the largeunfavorable entropy of immobilization that would result from peptidebinding. Thus, although the ribosome tunnel is made primarily of RNA,the nature of its surface is reminiscent of the interior surface of thechaperonin, GroEL (Xu et al. (1998) J. Struct. Biol. 124:129-141) in itsnon-binding conformation. Only in the conformation that exposes a largehydrophobic surface does GroEL bind a denatured protein.

There are six proteins (L19, L22, L23, L24, L29 and L31e) located at theexit from the tunnel, facing the translocon onto which the ribosomedocks during protein secretion. There is evidence that the ribosomebinds the translocon even after extensive digestion of its protein byprotease implying that interaction between the translocon and theribosome is mediated by RNA. The proximity of these proteins to thetranslocon, however, leads us to wonder what role, if any, they mightplay in the protein secretion process. Recent data from the Dobbersteinlaboratory shows that the N-terminal domain of SRP54, the G-protein fromthe signal recognition particle involved in signal peptide binding, canbe crosslinked to ribosomal proteins L23 and L29. These two proteins areadjacent to each other and at the tunnel exit (see FIG. 19).

7. Evolution.

In vitro evolution of RNA oligonucleotides has produced small RNAmolecules that can bind molecules like the Yarus inhibitor effectivelyor catalyze the peptidyl transfer reaction (Zhaug et al. (1998) Chem.Biol. 5: 539-553; Welch et al. (1997) supra). The sequence and secondarystructure of one of these selected RNAs is reminiscent of the peptidyltransferase loop in domain V of 23S rRNA (Zhaug et al. (1998) supra).The most striking similarity is a five nucleotide sequence that isidentical to a sequence in domain V that includes the catalytic A2486,G2482 and the buried phosphate of A2485. Remarkably, all of the groupsinvolved in the proposed charge relay system for activating A2486 in theribosome, are present in the in vitro selected ribozyme. Thus, thoughthe surrounding structural context is likely to be different, it seemsplausible that this artificially evolved ribozyme uses the samemechanisms as the ribosome for shifting the pKa of an adenine andlikewise uses it as a base for peptide synthesis. A second RNA (Welch etal. (1997) supra) contains a 12 nucleotide loop that includes a 9-basesequence identical to that found in the same region of the peptidyltransferase loop.

The striking similarities between the sequences containing the keycatalytic elements found in the peptidyl-transferase active site of theribosome and sequences of in vitro selected RNAs having relatedactivities make it clear that the appearance of a small RNA domaincapable of catalyzing peptidyl transferase was a plausible first step inthe evolution of protein synthesis on the ribosome. The first peptidessynthesized by this primordial peptide synthesizing enzyme might havebeen random polymers or copolymers, and it may have functioned withsubstrates as simple as an aminoacylated CCA. Basic peptides of thetypes observed to form the non-globular extensions that co-fold with the23S rRNA might have been among the first peptides synthesized that werefunctionally useful. Such peptides may have enhanced the stability ofthe protoribosome and other early ribozymes as the more sophisticatedpeptides of the present day ribosome appear to do.

C. Atomic Structure of the Large Ribosomal Subunit at 2.4 Å Resolution,Complete Refinement

The three-dimensional structure of the large ribosomal subunit fromHaloarcula marismortui has now been completely refined at 2.4 Åresolution. The model includes 2876 RNA nucleotides, 3701 amino acidsfrom 28 ribosomal proteins, 117 magnesium ions, 88 monovalent cations,and 7898 water molecules. Many of its proteins consist of asurface-exposed globular domain and one or more basic, non-globularextensions that are buried in rRNA. Half of them include motifs commonin non-ribosomal proteins including, for example: RRM domains, SH3-likebarrels and zinc fingers. Proteins that have significant sequence andstructural similarity, such as L15 and L18e, make essentially identicalinteractions with rRNA.

More particularly, the H. marismortui 50S subunit has been completelyrebuilt and refined by successive rounds of gradient energy minimizationand B-factor refinement using CNS (Brünger et al. (1998) supra).Ribosomal proteins and rRNA were completely rebuilt using the softwareprogram “0” (Jones, T. A. et al. (1991) Acta Crystallogr. A46: 110-119)with 2F_(o)-Fc electron density maps prior to the modeling of solventand metal ions. Modeling errors in the proteins were identified usingPROCHECK (Laskowski et al. (1993) J. Appl. Cryst. 26: 283-291) and byinspection of F_(o)-Fc maps. Difference maps also aided in theidentification of errors in the rRNA, most often associated with sugarpuckers. In the process, some adjustments were made in amino acidconformations, sequence register and in sequences themselves. Sequencechanges made were largely limited to L10e, L15e, and L37Ae, the onlythree proteins from the H. marismortui 50S that have not been sequenceddirectly. In addition, fifty-one amino acids were added to the modeldescribed in section IIA with forty-four of these coming from L10 at thebase of the L7/L12 stalk and L39e which lines a portion of the wall ofthe polypeptide exit tunnel. Fewer adjustments were made to the rRNAstructure. Forty-nine new nucleotides were modeled and refined, mainlyin helices 43 and 44 in domain II of 23S rRNA. In addition, the sugarpucker or conformation about the glycosidic bond was adjusted for somenucleotides. The refinement process was monitored by the quality ofelectron density maps calculated using phases derived from the model aswell as R/R_(free) values. The completely refined model now includes2876 RNA nucleotides, 3701 amino acids, 210 metal ions, and 7898 watermolecules. The model refines to an R/R_(free) of 18.9%/22.3% and hasexcellent geometry (Table 4).

Solvent modeling began with the generation of a list of possiblemagnesium ions obtained by an automatic peak selection using CNS. Peaksgreater than 3.5σ in F_(o)-Fc maps positioned within the magnesiuminner-sphere coordination distance of 1.9-2.1 Å from N or O atoms wereselected. The resulting list was manually inspected and only peaks thatdisplayed clear octahedral coordination geometry were selected asmagnesium.

Monovalent cations were identified on the basis of isomorphousdifferences between rubidium-soaked and native crystals of the H.marismortui 50S subunit. Since native crystals were stabilized in thepresence of 1.6M NaCl, these sites were initially modeled and refined asNa⁺¹. Refinement of these sites as K⁺¹ almost always resulted inunusually high temperature factors, with two exceptions where we havemodeled K⁺¹. Most of the monovalent sites in the 50S subunit appear tooccupied by Na⁺¹ in our crystals, however, these sites are likely to beoccupied by K⁺¹ in vivo.

Waters were selected as peaks greater than 3.5σ in F_(o)-Fc electrondensity maps and between 2.5 and 3.3 Å of O or N atoms. IndividualB-factor values were used to assess the assignment of water molecules. Anumber of waters refined to B-factors significantly lower thansurrounding RNA and protein atoms. In many cases these peaks were foundto be metal or chloride ions. A small number of low B-factor watermolecules were retained in the final model because they could not beunambiguously assigned as other species. As a result of adding metalions and water molecules the final model now contains 98, 547non-hydrogen atoms. The refinement and model statistics for the largeribosomal subunit are summarized in Table 4. TABLE 4 Refinement andModel Statistics for the H. marismortui 50S Subunit Space Group C222₁ a= 211.66 Å, b = 299.67 Å, c = 573.77 Å Total non-hydrogen atoms 98,542RNA atoms 61,617 Protein atoms 28,800 Water molecules 7,893 Magnesiumions 117 Potassium ions 2 Sodium ions 86 Chloride ions 22 Cadmium ions 5Refinement Statistics: Resolution Range 15.0-2.4 Å Number of reflectionsused in refinement 623,525 Number of reflections for cross-validation6,187 R_(working) 18.9% R_(free) 22.3% σ_(a) coordinate error(cross-validated) 0.35 Å (0.43 Å) luzzati coordinate error(cross-validated) 0.29 Å (0.35 Å) Deviations from ideality: r.m.s.d.bond lengths 0.0052 Å r.m.s.d. bond angles 1.13° r.m.s.d. dihedrals15.7° r.m.s.d. impropers 2.12° Protein Statistics from RamachandranPlot: Residues in most favored regions 2704 (86.6%) Residues inadditional allowed regions 379 (12.1%) Residues in generously allowedregions 27 (0.9%) Residues in disallowed regions 13 (0.4%) AverageB-factor Statistics (Å²): All atoms (high/low) 44.3 (10.1/133.7) rRNA41.2 (11.78/125.0) proteins 49.7 (13.9/92.5) waters 41.89 (9.58/115.4)

Refinement has also permitted additional modeling of L10, L39e, and theL11 binding site in 23S rRNA. Furthermore, it has been discovered thatcertain motifs, for example, RRM topologies, SH3-like barrels and zincfingers are common in the 50S proteins and each recognizes rRNA in manydifferent ways. Proteins that have significant three-dimensionalhomology, however, such as L15 and L18e as well as L18 and S11, makeessentially identical interactions with rRNA. Additional structuralhomologies between 50S proteins and non-ribosomal proteins also areapparent. The solvent exposed surfaces of these globular protein domainsare rich in aspartate and glutamate residues, while irregular proteinextensions penetrate the RNA core of the ribosome. These extensions areoften highly conserved, and their abundance of arginine, lysine, andglycine residues is important for their function. Collectively, theresults show evolutionary connections between many ribosomal proteinsand illustrate that protein-RNA interactions in the ribosome, althoughlargely idiosyncratic, share some common principles.

D. Antibiotic Binding Sites

In addition to the foregoing structural studies, the structure of thelarge ribosomal subunit of H. marismortui has been determined complexedwith each of seven different antibiotics. More specifically, crystals ofthe H. marismortui large ribosomal subunit have been soaked with one ofthe following antibiotics: anisomycin, blasticidin, carbomycin, tylosin,sparsomycin, virginiamycin or spiramycin. The structure of the largeribosome subunit complexed with each antibiotic was then resolved basedon X-ray diffraction data generated for each crystal.

Briefly, a small amount of a concentrated antibiotic solution was addedto a large subunit crystal suspended in stabilization solution andincubated for several hours. Following freezing and the other proceduresnormally used to prepare such crystals for experimental use, X-raydiffraction data were collected from the antibiotic containing crystals.Because the crystals were isomorphous to those from which the structuredescribed above was derived, the phases obtained for native crystal werecombined with the diffraction intensities obtained from theantibiotic-soaked crystal to obtain a structure for the latter. Theposition of the antibiotic in the crystal to which was bound is revealedmost clearly in difference electron density maps, which are electrondensity maps computed using the phases just referred to and amplitudesobtained by subtracting the amplitudes of crystals that contain noantibiotic from the (suitably scaled) amplitudes of those that containantibiotic. By using the foregoing methods, it was possible to determinethe atomic co-ordinates that show the spatial relationship betweenparticular antibiotics and their binding sites within the largeribosomal subunit. It is contemplated that similar methods can be usedto resolve the structure of other antibiotics complexed to the largeribosomal subunit.

The atomic co-ordinates of the large ribosomal subunit complexed withanisomycin are listed in a table on compact disk Disk No. 3 of 3 underthe file name anisomycin.pdb. In addition, FIG. 20 shows the spatialrelationship between the antibiotic anisomycin and the large ribosomalsubunit.

The atomic co-ordinates of the large ribosomal subunit complexed withblasticidin are listed in a table on compact Disk No. 3 of 3 under thefile name blasticidin.pdb. FIG. 21 shows the spatial relationshipbetween the antibiotic blasticidin and the large ribosomal subunit. Fororientation, FIG. 21 also includes a substrate for the P-site.

The atomic co-ordinates of the large ribosomal subunit complexed withcarbomycin are listed in a table on compact disk Disk No. 3 of 3 underthe file name carbomycin.pdb. FIG. 22 shows the spatial relationshipbetween the antibiotic carbomycin and the large ribosomal subunit. FIG.22 also shows a portion of the polypeptide exit tunnel.

The atomic co-ordinates of the large ribosomal subunit complexed withtylosin are listed in a table on compact disk Disk No. 3 of 3 under thefile name tylosin.pdb. FIG. 22 shows the spatial relationship betweenthe antibiotic tylosin and the large ribosomal subunit. FIG. 22 alsoshows a portion of the polypeptide exit tunnel.

The atomic co-ordinates of the large ribosomal subunit complexed withsparsomycin are listed in a table on compact disk Disk No. 3 of 3 underthe file name sparsomycin.pdb. FIG. 23 shows the spatial relationshipbetween the antibiotic sparsomycin and the large ribosomal subunit. Fororientation, FIG. 23 also shows a substrate for the P-site.

The atomic co-ordinates of the large ribosomal subunit complexed withvirginiamycin are listed in a table on compact disk Disk No. 3 of 3under the file name virginiamycin.pdb. FIG. 24 shows the spatialrelationship between the antibiotics virginiamycin as well carbomycin,and the large ribosomal subunit.

The atomic co-ordinates of the large ribosomal subunit complexed withspiramycin are listed in a table on compact disk Disk No. 3 of 3 underthe file name spiramycin.pdb.

FIG. 25 shows the spatial orientations of several antibiotics, namely,blasticidin, anisomycin, virginiamycin and carbomycin, as they bind totheir respective antibiotic binding sites within the large ribosomalsubunit. For purposes of orienting the reader, the positions of theP-site, A-site and the polypeptide exit tunnel are shown in FIG. 25. Asis apparent, these antibiotics bind to or contact specific locationswithin the large ribosomal subunit to disrupt protein biosynthesis. Forexample, it appears that blasticidin binds the large ribosomal subunitin the vicinity of the P-site; anisomycin and virginiamycin bind thelarge ribosomal subunit in the vicinity of the A-site; and carbomycin (amacrolide) binds the large ribosomal subunit in the vicinity of thepolypeptide exit tunnel adjacent the peptidyl transferase site.

From FIG. 25, it is apparent that the skilled artisan may identifycertain portions of each antibiotic that contact regions in the largeribosomal subunit. By knowing their spatial relationship with respectone another, the skilled artisan may generate a hybrid antibioticmolecule comprising a portion of a first template antibiotic and aportion of a second, different template antibiotic. The two portions maybe linked by a chemical linker so as to maintain the spatial orientationof one portion with respect to the other portion. As a result, thehybrid antibiotic may simultaneously bind each of the regions of theribosomal subunit typically bound by each template antibiotic. Thedesign and testing of such molecules is discussed in more detail below.

E. Experimental Techniques which Exploit X-Ray Diffraction Data

Based on the X-ray diffraction pattern obtained from the assemblage ofthe molecules or atoms in a crystalline solid, the electron density ofthat solid may be reconstructed using tools well known to those skilledin the art of crystallography and X-ray diffraction techniques.Additional phase information extracted either from the diffraction dataand available in the published literature and/or from supplementingexperiments may then used to complete the reconstruction.

For basic concepts and procedures of collecting, analyzing, andutilizing X-ray diffraction data for the construction of electrondensities see, for example, Campbell et al. (1984) BiologicalSpectroscopy, The Benjamin/Cummings Publishing Co., Inc., (Menlo Park,Calif.); Cantor et al. (1980) Biophysical Chemistry. Part II: Techniquesfor the study of biological structure and function, W.H. Freeman andCo., San Francisco, Calif.; A. T. Brünger (1993) X-PLOR Version 3.1: Asystem for X-ray crystallography and NMR, Yale Univ. Pr., (New Haven,Conn.); M. M. Woolfson (1997) An Introduction to X-ray Crystallography,Cambridge Univ. Pr., (Cambridge, UK); J. Drenth (1999) Principles ofProtein X-ray Crystallography (Springer Advanced Texts in Chemistry),Springer Verlag; Berlin; Tsirelson et al. (1996) Electron Density andBonding in Crystals: Principles Theory and X-ray Diffraction Experimentsin Solid State Physics and Chemistry, Inst. of Physics Pub.; U.S. Pat.No. 5,942,428; U.S. Pat. No. 6,037,117; U.S. Pat. No. 5,200,910 and U.S.Pat. No. 5,365,456 (“Method for Modeling the Electron Density of aCrystal”).

A molecular model may then be progressively built using the experimentalelectron density information and further refined against the X-raydiffraction data resulting in an accurate molecular structure of thesolid.

F. Structural Determination of Other Large Ribosomal Subunits

It is understood that the skilled artisan, when provided with the atomicco-ordinates of a first macromolecule may use this information toquickly and easily determine the three-dimensional structure of adifferent but structurally related macromolecule. For example, theatomic co-ordinates defining the large ribosomal subunit from H.marismortui can be used to determine the structure of the largeribosomal subunit from other species either as an isolated subunit, incomplex with the small subunit, or either of these complexed withfunctionally important ligands, for example: aminoacyl tRNA; variousprotein synthesis factors, such as elongation factor G, elongationfactor Tu, termination factor or recycling factor, in both their GTP andGDP conformational states; and protein synthesis inhibitors, forexample, antibiotics. In addition, the H. marismortui subunitco-ordinates can also be used to solve the structures of ribosomalcomplexes with components of the protein secretion machinery, forexample, the signal recognition particle, and the translocon.

If the crystal being examined contains a macromolecule of unknownstructure and no additional information is available, additionalexperiments sometimes may be required to determine the relevant phasesof the macromolecule. These studies can often be time consuming anduncertain of success (Blundell et al. (1976) supra). However, whenadditional information, for example, structural and/or crystallographicinformation, is available for molecules related in some way to themacromolecule of interest then the process of resolving the structure ofthe molecule of interest is a much less challenging and time-consumingtask.

Accordingly, the skilled artisan may use information gleaned from theprior resolved structure to develop a three-dimensional model of a newmolecule of interest. Furthermore, the skilled artisan may use a varietyof approaches to elucidate the three-dimensional structure of the newmolecule. The approaches may depend on whether crystals of the moleculeof interest are available and/or whether the molecule of interest has ahomologue whose structure has already been determined.

In one approach, if the molecule of interest forms crystals that areisomorphous, i.e., that have the same unit cell dimensions and spacegroup as a related molecule whose structure has been determined, thenthe phases and/or co-ordinates for the related molecule can be combineddirectly with newly observed amplitudes to obtain electron density mapsand, consequently, atomic co-ordinates of the molecule of interest. Theresulting maps and/or atomic co-ordinates may then be refined usingstandard refinement techniques known in the art. In another approach, ifthe molecule of interest is related to another molecule of knownthree-dimensional structure, but crystallizes in a different unit cellwith different symmetry, the skilled artisan may use a technique knownas molecular replacement to obtain useful phases from the co-ordinatesof the molecule whose structure is known (Blundell et al. (1976) supra).These phases can then be used to generate an electron density map and/oratomic co-ordinates for the molecule of interest. In another approach,if no crystals are available for the molecule of interest but it ishomologous to another molecule whose three-dimensional structure isknown, the skilled artisan may use a process known as homology modelingto produce a three-dimensional model of the molecule of interest. It iscontemplated that other approaches may be useful in deriving athree-dimensional model of a molecule of interest. Accordingly,information concerning the crystals and/or atomic co-ordinates of onemolecule can greatly facilitate the determination of the structures ofrelated molecules.

The method of molecular replacement, developed initially by Rossmann andBlow in the 1960s, is now used routinely to establish the crystalstructures of macromolecules of unknown structures using the structureof a homologous molecule, or one in a different state of ligation (M. G.Rossmann, ed. “The Molecular Replacement Methods,” Int. Sci. Rev. J. No.13, Gordon & Breach, New York, N.Y. (1972); Eaton Lattman, “Use ofRotation and Translation Functions,” H. W. Wyckoff, C. H. W. Hist. (S.N. Timasheff, ed.) Methods in Enzymology, 115: 55-77 (1985)). For anexample of the application of molecular replacement, see, for example,Rice, P. A. & Steitz, T. A. (1994) EMBO J. 13: 1514-24.

In molecular replacement, the three-dimensional structure of the knownmolecule is positioned within the unit cell of the new crystal byfinding the orientation and position that provides the best agreementbetween observed diffraction amplitudes and those calculated from theco-ordinates of the positioned subunit. From this modeling, approximatephases for the unknown crystal can be derived. In order to position aknown structure in the unit cells of an unknown, but related structure,three rotation angles and three translations relative to the unit cellorigin have to be determined. The rotation search is carried out bylooking for agreement between the Patterson function of the search andtarget structures as a function of their relative orientation (therotation function). X-PLOR (Brünger et al. (1987) Science 235:458-460;CNS (Crystallography & NMR System, Brünger et al., (1998) Acta Cryst.Sect. D 54: 905-921), and AMORE: an Automatic Package for MolecularReplacement (Navaza, J. (1994) Acta Cryst. Sect. A, 50: 157-163) arecomputer programs that can execute rotation and translation functionsearches. Once the orientation of a test molecule is known, the positionof the molecule must be found using a translational search. Once theknown structure has been positioned in the unit cell of the unknownmolecules, phases for the observed diffraction data can be calculatedfrom the atomic co-ordinates of the structurally related atoms of theknown molecules. By using the calculated phases and X-ray diffractiondata for the unknown molecule, the skilled artisan can generate anelectron density map and/or atomic co-ordinates of the molecule ofinterest.

By way of example, it is contemplated that a three-dimensional model ofa ribosomal subunit other than that derived from H. marismortui can begenerated via molecular replacement. In this method, the H. marismortuisubunit structures are positioned within the unit cell of the newcrystal by finding the orientation and position that provides the bestagreement between observed diffraction amplitudes and those calculatedfrom the co-ordinates of the positioned subunit. A starting electrondensity map calculated using 2F_(hkl)(observed)-F_(hkl)(calculated),where F(observed) are the diffraction amplitudes that have been measuredfrom crystals of the unknown structure, and F(calculated) are thediffraction amplitudes calculated from the positioned H. marismortuisubunit structure. Refinement of the initial model can be done as isstandard in the field of macromolecular crystallography.

The H. marismortui 50S structure can also be used to establish thestructure of a 70S ribosome or 50S ribosome for which an electiondensity map has been calculated, at a resolution that would otherwise betoo low to be interpreted, while a 5 Å resolution map could not beinterpreted in atomic terms de novo, a plausible atomic model can beconstructed by refitting the H. marismortui 50S structure to a lowerresolution map (e.g., 4.5 Å to 8 Å). This refitting can be combined withhomology modeling to obtain a three-dimensional model of a ribosome orribosomal subunit from a different species. It is contemplated thatsimilar procedures may be used to determine the structure of theeukaryotic 60S subunit and/or a eukaryotic ribosome.

In general, the success of molecular replacement for solving structuresdepends on the fraction of the structures that are related and theirdegree of identity. For example, if about 50% or more of the structureshows an r.m.s. difference between corresponding atoms in the range ofabout 2 Å or less, the known structure can be successfully used to solvethe unknown structure.

Homology modeling, also known as comparative modeling or knowledge-basedmodeling, can be used to generate a three-dimensional model for amolecule based on the known structure of homologues. In general, theprocedure may comprise one or more of the following steps: aligning theamino acid or nucleic acid sequence of an unknown molecule against theamino acid or nucleic acid sequence of a molecule whose structure haspreviously been determined; identifying structurally conserved andstructurally variable regions; generating atomic co-ordinates for core(structurally conserved) residues of the unknown structure from those ofthe known structure(s); generating conformations for the other(structurally variable) residues in the unknown structure; building sidechain conformations; and refining and/or evaluating the unknownstructure.

By way of example, since the nucleotide sequences of all known 50Ssubunit rRNAs can be aligned relative to each other and to H.marismortui 23S and 5S rRNAs, it is possible to construct models of thestructures of other 50S ribosomal rRNAs, particularly in the regions ofthe tunnel and active sites, using the H. marismortui structure.Likewise, homologous proteins can also be modeled using similarmethodologies. Methods useful for comparative RNA sequence analysis areknown in the art and include visual methods and number pattern methods,as well as methods employing chi-square statistics, phylogeneticalgorithms, or empirical algorithms. Descriptions of some of theforegoing methods are available, for example, athttp://www.rna.icmb.utexas.edu/; Gutell (1996), “Comparative SequenceAnalysis and the Structure of 16S and 23S rRNA,” Ribosomal RNA.Structure, Evolution, Processing, and Function in Protein Biosynthesis,(Dahlberg A. and Zimmerman B., eds.) CRC Press. Boca Raton, pp. 111-128;Guttell et al. (1993) Nucl. Acid Res. 21: 3055-3074; Schnare et al.(1996) J. Mol. Biol. 256: 701-719. Particularly useful visual inspectionmethods include comparison of a particular position in a H. marismortuisecondary structure diagram with the residues located at the analogousposition on an E. coli secondary structure diagram. A software programthat is particularly useful in homology modeling includes XALIGN(Wishart, D. et al., (1994) Cabios 10: 687-88). See also, U.S. Pat. No.5,884,230.

To model the rRNA of a new species, bases of the H. marismortui rRNA canbe replaced, using a computer graphics program such as “O” (Jones etal., (1991) Acta Cryst. Sect. A, 47: 110-119), by those of thehomologous rRNA, where they differ. In many if not most cases the sameorientation of the base will be appropriate. Insertions and deletionsmay be more difficult and speculative, but the rRNA forming the peptidyltransferase site and the portion of the tunnel closest to it is veryhighly conserved with essentially no insertions and deletions. Automatedweb-based homology modeling can be performed using, for example, thecomputer programs SWISS-MODEL available through Glaxo WellcomeExperimental Research in Geneva, Switzerland, and WHATIF available onEMBL servers.

For other descriptions of homology modeling, see, for example, Gutell R.R. (1996), supra; Gutell R. R., et al. (1993) Nucleic Acids Res.21:3055-3074; Schnare et al. (1996) J. Mol. Biol., 256: 701-719;Blundell et al. (1987) Nature 326: 347-352; Fetrow and Bryant (1993)Bio/Technology 11:479-484; Greer (1991) Methods in Enzymology 202:239-252; and Johnson et al. (1994) Crit. Rev. Biochem. Mol. Biol.29:1-68. An example of homology modeling can be found, for example, inSzklarz G. D (1997) Life Sci. 61: 2507-2520.

As discussed earlier, the large ribosomal subunit from prokaryotes andeukaryotes are structurally conserved. The amino acid sequences of thelarge ribosomal subunit from prokaryotes and eukaryotes can be aligneddue to the evolutionary conservation of amino acid residues that areimportant for three-dimensional structure, the nature and shape of thebinding sites for substrates and the catalytic site. This similarity inamino acid sequence of the homologous large ribosomal subunit allows theconstruction of models, via homology modeling, for the molecules whosecrystal structures have not been solved.

The new ribosome or large ribosomal subunit structures determined usingthe H. marismortui crystals and/or atomic co-ordinates can then be usedfor structure-based drug design using one or more of the approachesdescribed hereinbelow. This information can then be used to designmolecules that selectively bind and disrupt protein synthesis in theribosomes of the pathogens while leaving the ribosomes of a hostrelatively unaffected.

G. Rational Drug Design

1. Introduction

It is contemplated that the atomic co-ordinates defining a largeribosomal subunit of interest, whether derived from one or more of X-raycrystallography, molecular modeling, homology modeling or molecularreplacement, may be used in rational drug design (RDD) to design a novelmolecule of interest, for example, novel modulators (for example,inducers, mimetics or inhibitors) of ribosome function. Furthermore, itis contemplated that, by using the principles disclosed herein, theskilled artisan can design, make, test, refine and use novel proteinsynthesis inhibitors specifically engineered to reduce, disrupt, orotherwise or inhibit ribosomal function in an organism or species ofinterest. For example, by using the principles discussed herein, theskilled artisan can engineer new molecules that specifically target andinhibit ribosomal function in a pathogen, for example, a particularprokaryotic, organism, while preserving ribosomal function in a host,for example, a eukaryotic organism, specifically a mammal, and morespecifically, a human. As a result, the atomic co-ordinates provided anddiscussed herein permit the skilled artisan to design new antibioticsthat can kill certain pathogenic organisms while having little or notoxicity in the intended recipient, for example, a human.

It is contemplated that RDD using atomic co-ordinates of the largeribosomal subunit can be facilitated most readily via computer-assisteddrug design (CADD) using conventional computer hardware and softwareknown and used in the art. The candidate molecules may be designed denovo or may be designed as a modified version of an already existingmolecule, for example, a pre-existing antibiotic, using conventionalmethodologies. Once designed, candidate molecules can be synthesizedusing standard methodologies known and used in the art. Followingsynthesis, the candidate molecules can be screened for bioactivity, forexample, by their ability to reduce or inhibit ribosome function, theirability to interact with or bind a ribosome or a ribosomal subunit.Based in part upon these results, the candidate molecules may be refinediteratively using one or more of the foregoing steps to produce a moredesirable molecule with a desired biological activity. The resultingmolecules can be useful in treating, inhibiting or preventing thebiological activities of target organisms, thereby killing the organismor impeding its growth. Alternatively, the resulting molecules can beuseful for treating, inhibiting or preventing microbial infections inany organism, particularly animals, more particularly humans.

In summary, the tools and methodologies provided by the presentinvention may be used to identify and/or design molecules which bindand/or interact in desirable ways with ribosomes and ribosomal subunits.Basically, the procedures utilize an iterative process whereby themolecules are synthesized, tested and characterized. New molecules canbe designed based on the information gained in the testing andcharacterization of the initial molecules and then such newly identifiedmolecules can themselves be tested and characterized. This series ofprocesses may be repeated as many times as necessary to obtain moleculeswith desirable binding properties and/or biological activities. Methodsfor identifying candidate molecules are discussed in more detail below.

2. Identification of Candidate Molecules

It is contemplated that the design of candidate molecules of interestcan be facilitated by conventional ball and stick-type modelingprocedures. However, in view of the size and complexity of the largeribosomal subunit, it is contemplated that the ability to designcandidate molecules may be enhanced significantly using computer-basedmodeling and design protocols.

a. Molecular Modeling.

It is contemplated that the design of candidate molecules, as discussedin detail hereinbelow, can be facilitated using conventional computersor workstations, available commercially from, for example, SiliconGraphics Inc. and Sun Microsystems, running, for example, UNIX based,Windows NT on IBM OS/2 operating systems, and capable of runningconventional computer programs for molecular modeling and rational drugdesign.

It is understood that any computer system having the overallcharacteristics set forth in FIG. 27 may be useful in the practice ofthe invention. More specifically, FIG. 27, is a schematic representationof a typical computer work station having in electrical communication(100) with one another via, for example, an internal bus or externalnetwork, a central processing unit (101), a random access memory (RAM)(102), a read only memory (ROM) (103), a monitor or terminal (104), andoptimally an external storage device, for example, a diskette, CD ROM,or magnetic tape (105).

The computer-based systems of the invention preferably comprise a datastorage means having stored therein a ribosome or ribosomal subunit orfragment sequence and/or atomic co-ordinate/X-ray diffraction data ofthe present invention and the necessary hardware means and softwaremeans for supporting and implementing an analysis means. As used herein,“a computer system” or “a computer-based system” refers to the hardwaremeans, software means, and data storage means used to analyze thesequence, X-ray diffraction data, and/or atomic co-ordinates of theinvention. As used herein, the term “data storage means” is understoodto refer to any memory which can store sequence data, atomicco-ordinates, and/or X-ray diffraction data, or a memory access meanswhich can access manufactures having recorded thereon the atomicco-ordinates of the present invention.

In one embodiment, a ribosome or ribosomal subunit, or at least asubdomain thereof, amino acid and nucleic acid sequence, X-raydiffraction data and/or atomic co-ordinates of the present invention arerecorded on computer readable medium. As used herein, the term “computerreadable medium” is understood to mean any medium which can be read andaccessed directly by a computer. Such media include, but are not limitedto: magnetic storage media, such as floppy discs, hard disc storagemedium, and magnetic tape; optical storage media such as optical discsor CD-ROM; electrical storage media such as RAM and ROM; and hybrids ofthese categories such as magnetic/optical storage media. A skilledartisan can readily appreciate how any of the presently known computerreadable mediums can be used to create a manufacture comprising computerreadable medium having recorded thereon an amino acid and/or nucleotidesequence, X-ray diffraction data, and/or atomic co-ordinates of thepresent invention.

As used herein, the term “recorded” is understood to mean any processfor storing information on computer readable medium. A skilled arts ancan readily adopt any of the presently known methods for recordinginformation on computer readable medium to generate manufacturescomprising an amino acid or nucleotide sequence, atomic co-ordinatesand/or X-ray diffraction data of the present invention.

A variety of data storage structures are available to a skilled artisanfor creating a computer readable medium having recorded thereon aminoacid and/or nucleotide sequence, atomic co-ordinates and/or X-raydiffraction data of the present invention. The choice of the datastorage structure will generally be based on the means chosen to accessthe stored information. In addition, a variety of data processorprograms and formats can be used to store the sequence information,X-ray data and/or atomic co-ordinates of the present invention oncomputer readable medium. The foregoing information, data andco-ordinates can be represented in a word processing text file,formatted in commercially-available software such as WordPerfect andMICROSOFT Word, or represented in the form of an ASCII file, stored in adatabase application, such as DB2, Sybase, Oracle, or the like. Askilled artisan can readily adapt any number of data processorstructuring formats (e.g. text file or database) in order to obtaincomputer readable medium having recorded thereon the information of thepresent invention.

By providing a computer readable medium having stored thereon a ribosomeor ribosomal subunit sequence, and/or atomic co-ordinates, a skilledartisan can routinely access the sequence, and/or atomic co-ordinates tomodel a ribosome or ribosomal subunit, a subdomain thereof, mimetic, ora ligand thereof. Computer algorithms are publicly and commerciallyavailable which allow a skilled artisan to access this data provided ina computer readable medium and analyze it for molecular modeling and/orRDD. See, e.g., Biotechnology Software Directory, MaryAnn Liebert Publ.,New York, N.Y. (1995).

Although computers are not required, molecular modeling can be mostreadily facilitated by using computers to build realistic models of aribosome, ribosomal subunit, or a portion thereof. Molecular modelingalso permits the modeling of new smaller molecules, for example ligands,agents and other molecules, that can bind to a ribosome, ribosomalsubunit, or a portion therein. The methods utilized in molecularmodeling range from molecular graphics (i.e., three-dimensionalrepresentations) to computational chemistry (i.e., calculations of thephysical and chemical properties) to make predictions about the bindingof the smaller molecules or their activities; to design new molecules;and to predict novel molecules, including ligands such as drugs, forchemical synthesis.

For basic information on molecular modeling, see, for example, M.Schlecht, Molecular Modeling on the PC (1998) John Wiley & Sons; Gans etal., Fundamental Principals of Molecular Modeling (1996) Plenum Pub.Corp.; N. C. Cohen, ed., Guidebook on Molecular Modeling in Drug Design(1996) Academic Press; and W. B. Smith, Introduction to TheoreticalOrganic Chemistry and Molecular Modeling (1996). U.S. patents whichprovide detailed information on molecular modeling include, for example:U.S. Pat. Nos. 6,093,573; 6,080,576; 6,075,014; 6,075,123; 6,071,700;5,994,503; 5,884,230; 5,612,894; 5,583,973; 5,030,103; 4,906,122; and4,812,12.

Three-dimensional modeling can include, but is not limited to, makingthree-dimensional representations of structures, drawing pictures ofstructures, building physical models of structures, and determining thestructures of related ribosomes, ribosomal subunits and ribosome/ligandand ribosomal subunit/ligand complexes using the known co-ordinates. Theappropriate co-ordinates are entered into one or more computer programsfor molecular modeling, as known in the art. By way of illustration, alist of computer programs useful for viewing or manipulatingthree-dimensional structures include: Midas (University of California,San Francisco); MidasPlus (University of California, San Francisco);MOIL (University of Illinois); Yummie (Yale University); Sybyl (Tripos,Inc.); Insight/Discover (Biosym Technologies); MacroModel (ColumbiaUniversity); Quanta (Molecular Simulations, Inc.); Cerius (MolecularSimulations, Inc.); Alchemy (Tripos, Inc.); LabVision (Tripos, Inc.);Rasmol (Glaxo Research and Development); Ribbon (University of Alabama);NAOMI (Oxford University); Explorer Eyechem (Silicon Graphics, Inc.);Univision (Cray Research); Molscript (Uppsala University); Chem-3D(Cambridge Scientific); Chain (Baylor College of Medicine); O (UppsalaUniversity); GRASP (Columbia University); X-Plor (Molecular Simulations,Inc.; Yale University); Spartan (Wavefunction, Inc.); Catalyst(Molecular Simulations, Inc.); Molcadd (Tripos, Inc.); VMD (Universityof Illinois/Beckman Institute); Sculpt (Interactive Simulations, Inc.);Procheck (Brookhaven National Library); DGEOM (QCPE); RE_VIEW (BrunellUniversity); Modeller (Birbeck College, University of London); Xmol(Minnesota Supercomputing Center); Protein Expert (CambridgeScientific); HyperChem (Hypercube); MD Display (University ofWashington); PKB (National Center for Biotechnology Information, NIH);ChemX (Chemical Design, Ltd.); Cameleon (Oxford Molecular, Inc.); andIditis (Oxford Molecular, Inc.).

One approach to RDD is to search for known molecular structures thatmight bind to a site of interest. Using molecular modeling, RDD programscan look at a range of different molecular structures of molecules thatmay fit into a site of interest, and by moving them on the computerscreen or via computation it can be decided which structures actuallyfit the site well (William Bains (1998) Biotechnology from A to Z,second edition, Oxford University Press, p. 259).

An alternative but related approach starts with the known structure of acomplex with a small molecule ligand and models modifications of thatsmall molecule in an effort to make additional favorable interactionswith a ribosome or ribosomal subunit.

The present invention permits the use of molecular and computer modelingtechniques to design and select novel molecules, such as antibiotics orother therapeutic agents, that interact with ribosomes and ribosomalsubunits. Such antibiotics and other types of therapeutic agentsinclude, but are not limited to, antifungals, antivirals,antibacterials, insecticides, herbicides, miticides, rodentcides, etc.

In order to facilitate molecular modeling and/or RDD the skilled artisanmay use some or all of the atomic co-ordinates deposited at the RCSBProtein Data Bank with the accession numbers PDB ID: 1FFK, 1JJ2, 1FFZ,or 1FG0, and/or those atomic co-ordinates contained on Disk No. 1, 2 or3 of 3. Furthermore, the skilled artisan, using the foregoing atomicco-ordinates, the skilled artisan can generate additional atomicco-ordinates via, for example, molecular modeling using, for example,homology modeling and/or molecular replacement techniques, that togetherdefine at least a portion of a model of a ribosome from another speciesof interest. By using the foregoing atomic co-ordinates, the skilledartisan can design inhibitors of protein synthesis that may be tailoredto be effective against ribosomes from one or more species but whichhave little or no effect on ribosomes of other species. Such inhibitorsmay be competitive inhibitors. As used herein, the term “competitiveinhibitor” refers to an inhibitor that binds to the active form of aribosome or ribosomal subunit at the same sites as its substrate(s) ortRNA(s), thus directly competing with them. The term “active form” of aribosome or ribosomal subunit refers to a ribosome or ribosomal subunitin a state that renders it capable of protein synthesis. Competitiveinhibition can be reversed completely by increasing the substrate ortRNA concentration.

This invention also permits the design of molecules that act asuncompetitive inhibitors of protein synthesis. As used herein, the term“uncompetitive inhibitor” refers to a molecule that inhibits thefunctional activity of a ribosome or ribosomal subunit by binding to adifferent site on the ribosome or ribosomal subunit than does itssubstrates, or tRNA. Such inhibitors can often bind to the ribosome orribosomal subunit with the substrate or tRNA and not to the ribosome orribosomal subunit by itself. Uncompetitive inhibition cannot be reversedcompletely by increasing the substrate concentration. These inhibitorsmay bind to, all or a portion of, the active sites or other regions ofthe large ribosomal subunit already bound to its substrate and may bemore potent and less non-specific than known competitive inhibitors thatcompete for large ribosomal subunit active sites or for binding to largeribosomal subunit.

Similarly, non-competitive inhibitors that bind to and inhibit proteinsynthesis whether or not it is bound to another chemical entity may bedesigned using the atomic co-ordinates of the large ribosomal subunitsor complexes comprising large ribosomal subunit of this invention. Asused herein, the term “non-competitive inhibitor” refers to an inhibitorthat can bind to either the free or substrate or tRNA bound form of theribosome or ribosomal subunit.

Those of skill in the art may identify inhibitors as competitive,uncompetitive, or non-competitive by computer fitting enzyme kineticdata using standard equation according to Segel, I. H., (1975) EnzymeKinetics: Behaviour and Analysis of Rapid Equilibrium and Steady-StateEnzyme Systems, (Wiley Classics Library). It should also be understoodthat uncompetitive or non-competitive inhibitors according to thepresent invention may bind the same or different binding sites.

Alternatively, the atomic co-ordinates provided by the present inventionare useful in designing improved analogues of known protein synthesisinhibitors or to design novel classes of inhibitors based on the atomicstructures and co-ordinates of the crystals of the 50S ribosomalsubunit/CCdA-p-Puro complex and the 50S ribosomal subunit/aa-tRNAanalogue complex. This provides a novel route for designing inhibitorsof protein synthesis with both high specificity, stability and otherdrug-like qualities (Lipinski et al. (1997) Adv. Drug Deliv. Rev. 23:3).

The atomic co-ordinates of the present invention also permit probing thethree-dimensional structure of a ribosome or ribosome subunit or aportion thereof with molecules composed of a variety of differentchemical features to determine optimal sites for interaction betweencandidate inhibitors and/or activators and the ribosome or ribosomalsubunit. For example, high resolution atomic co-ordinates based on X-raydiffraction data collected from crystals saturated with solvent allowsthe determination of where each type of solvent molecule sticks. Smallmolecules that bind to those sites can then be designed and synthesizedand tested for their inhibitory activity (Travis, J. (1993) Science 262:1374). Further, any known antibiotic, inhibitor or other small moleculethat binds to the H. marismortui large subunit can be soaked into H.marismortui large subunit crystals and their exact mode of bindingdetermined from difference electron density maps. These molecules mayrepresent lead compounds from which better drug-like compounds can besynthesized.

b. Identification of Target Sites.

The atomic co-ordinates of the invention permit the skilled artisan toidentify target locations in a ribosome or large ribosomal subunit thatcan serve as a starting point in rational drug design. As a thresholdmatter, the atomic co-ordinates of the invention permit the skilledartisan to identify specific regions within a ribosome or ribosomalsubunit that are involved with protein synthesis and/or proteinsecretion out of the ribosome. Furthermore, the atomic co-ordinates ofthe invention permit a skilled artisan to further identify portions ofthese regions that are conserved or are not conserved between differentorganisms. For example, by identifying portions of these regions thatare conserved among certain pathogens, for example, certain prokaryotes,but are not conserved in a host organism, for example, a eukaryote, morepreferably a mammal, the skilled artisan can design molecules thatselectively inhibit or disrupt protein synthesis activity of thepathogen's but not the host's ribosomes. Furthermore, by analyzingregions that are either conserved or non-conserved between certainpathogens, it may be possible to design broad or narrow spectrum proteinsynthesis inhibitors, e.g., antibiotics, as a particular necessityarises.

FIG. 28, is a schematic representation of a large ribosomal subunit thatidentifies a variety of exemplary target sites that appear toparticipate in protein synthesis within the ribosome and/or the exportor translocation of the newly synthesized protein out of the ribosome.The target sites include, for example, the P-site (200), the A-site(201), the peptidyl transferase center (202), the peptidyl transferasesite (203) which includes at least a portion of the P-site and theA-site, a factor binding domain (204) including, for example, the EF-Tubinding domain and the EF-G binding domain, the polypeptide exit tunnel(205) including cavities defined by the wall of the exit tunnel, and thesignal recognition particle binding domain (206).

By way of example, inspection of the atomic co-ordinates of the H.marismortui 50S ribosomal subunit has identified a variety of targetregions that may serve as a basis for the rational drug design of new ormodified protein synthesis inhibitors. The target regions include thepeptidyl transferase site, A-site, the P-site, the polypeptide exittunnel, certain cavities disposed in the wall of the polypeptide exittunnel (for example, cavity 1 and cavity 2), and certain antibioticbinding pockets. The residues that together define at least a portion ofeach of the foregoing regions are identified in the following tables.However, it is contemplated that the same or similar target sites can beidentified in a ribosome or a ribosomal unit of interest using theprinciples described herein. Furthermore, these principles can beemployed using any of the primary sets of atomic co-ordinates providedherein or any additional atomic co-ordinate sets, for example, secondaryatomic co-ordinate sets that may be generated by molecular modeling ofany ribosome or ribosomal subunit of interest.

Table 5 identifies the residues in the H. marismortui SOS ribosomalsubunit that together define at least a portion of the ribosomalpeptidyl transferase site. In addition, Table 5 identifies which ofthose residues that define at least a portion of the peptidyltransferase site are not conserved between H. marismortui and E. coli,those that are not conserved between H. marismortui and rat, those thatare not conserved between E. coli and rat, and those that are notconserved between eubacteria and eukaryota. The non-conserved residueswere identified by comparison of sequences of H. marismortui 23S rRNAthat form the above-mentioned sites with the corresponding sequences ofaligned rRNA from the other organisms. TABLE 5 Residues that Define theRibosomal Peptidyl-Transferase Site Residues Not Residues Not ResiduesNot Conserved between Conserved Between Conserved Between ResidueConserved H. marismortui H. marismortui H. marismortui E. Coli andRattus Between Bacteria A-site Residues and E. coli and Rattus(Coli/Rat) and Eukaryotes? G2102 Yes A2103 Yes C2104 Yes C2105 Yes C2106Yes G2482 Yes G2284 Yes G2285 Yes G2286 Yes A2474 Yes A2485 Yes A2486Yes C2487 Yes A2488 U A/U No U2528 Yes C2536 Yes A2538 Yes G2540 YesU2541 Yes C2542 Yes G2543 Yes G2588 Yes U2589 Yes U2590 Yes C2608 YesG2617 Yes G2618 Yes U2619 Yes U2620 Yes A2635 Yes C2636 Yes A2637 YesG2638 YesResidues were determined by a 5.8 angstrom distance measurement betweenthe atoms of the CCdA-p-Puromycin A-site ligand and CCdA-Puromycintransition state inhibitor (PDB accession codes: 1fg0 and 1ffz,respectively) and the 50S ribosome using the program SPOCK^(a).Conserved residues were determined by comparison between the proposedsecondary structures of H. marismortui ^(b), E. coli ^(b), and Rattusnorvegicus ^(c).^(a)Christopher, Jon A. (1998) SPOCK: The Structural PropertiesObservation and Calculation Kit (Program Manual), The Center forMacromolecular Design, Texas A&M University, College Station, TX.^(b)Comparative RNA Web Site URL: http://www.rna.icmb.utexas.edu/^(c)Wool, I. G. (1985). “Studies of the Structure of Eukaryotic(Mammalian) Ribosomes” in Structure, Function, and Genetics ofRibosomes, supra, pp. 391-411.

Table 6 identifies the residues in the H. marismortui 50S ribosomalsubunit that together define at least a portion of the ribosomal A-site.In addition, Table 6 identifies which of those residues that define atleast a portion the A-site are not conserved between H. marismortui andE. coli, those that are not conserved between H. marismortui and rat,those that are not conserved between E. coli and rat, and those that arenot conserved between eubacteria and eukaryota. The non conservedresidues were identified as described previously with respect to Table5. TABLE 6 Residues that Define the Ribosomal A-site Residues NotResidues Not Residues Not Conserved between Conserved Between ConservedBetween Residue Conserved H. marismortui H. marismortui H. marismortuiE. Coli and Rattus Between Bacteria A-site Residues and E. coli andRattus (Coli/Rat) and Eukaryotes? G2102 Yes A2103 Yes C2104 Yes G2482Yes A2485 Yes A2486 Yes C2487 Yes A2488 U A/U No U2528 Yes C2536 YesA2538 Yes G2540 Yes U2541 Yes C2542 Yes G2543 Yes G2588 Yes U2589 YesU2590 Yes C2608 Yes G2617 Yes G2618 Yes U2619 Yes U2620 Yes A2637 YesResidues were determined by a 5.8 angstrom distance measurement betweenthe atoms of the CC-Puromycin A-site ligand and the 50S ribosome (PDBaccession codes: 1fg0) using the program SPOCK^(a). Conserved residueswere determined by comparison between the proposed secondary structuresof H. marismortui ^(b), E. coli ^(b), and Rattus norvegicus ^(c).^(a)Christopher, Jon A. (1998) SPOCK: The Structural PropertiesObservation and Calculation Kit (Program Manual), The Center forMacromolecular Design, Texas A&M University, College Station, TX.^(b)Comparative RNA Web Site URL: http://www.rna.icmb.utexas.edu/^(c)Wool, I. G. (1985). “Studies of the Structure of Eukaryotic(Mammalian) Ribosomes” in Structure, Function, and Genetics ofRibosomes, supra, pp. 391-411.

Table 7 identifies the residues in the H. marismortui 50S ribosomalsubunit that together define at least a portion of the ribosomal P-site.As demonstrated in Table 7, all of the residues in that portion of theribosomal P-sites are conserved between H. marismortui and E. colii,between H. marismortui and rat, between E. colii and rat, and betweeneubacteria and eukaryota, as determined using the comparison methoddescribed previously with respect to Table 5. TABLE 7 Residues thatDefine the Ribosomal P-site Residues Not Residues Not Residues NotConserved between Conserved Between Conserved Between Residue ConservedH. marismortui H. marismortui H. marismortui E. Coli and Rattus BetweenBacteria P-site Residues and E. coli and Rattus (Coli/Rat) andEukaryotes? C2104 Yes C2105 Yes C2106 Yes G2284 Yes G2285 Yes G2286 YesA2474 Yes A2485 Yes A2486 Yes U2619 Yes U2620 Yes A2635 Yes C2636 YesA2637 Yes G2638 YesResidues were determined by a 5.8 angstrom distance measurement betweenthe atoms of the CCA-PO₂ moiety of the CCdA-p-puromycin transition stateinhibitor and the 50S ribosome (PDB accession codes 1ffz) using theprogram SPOCK^(a). Conserved residues were determined by comparisonbetween the proposed secondary structures of H. marismortui ^(b), E.coli ^(b), and Rattus norvegicus ^(c).^(a)Christopher, Jon A. (1998) SPOCK: The Structural PropertiesObservation and Calculation Kit (Program Manual), The Center forMacromolecular Design, Texas A&M University, College Station, TX.^(b)Comparative RNA Web Site URL: http://www.rna.icmb.utexas.edu/^(c)Wool, I. G. (1985). “Studies of the Structure of Eukaryotic(Mammalian) Ribosomes” in Structure, Function, and Genetics ofRibosomes, supra, pp. 391-411.

Table 8 identifies the residues in the H. marismortuiSOS ribosomalsubunit that together define at least a portion of the ribosomalpolypeptide exit tunnel. In addition, Table 8 identifies which of thoseresidues that define at least a portion of the polypeptide exit tunnelare not conserved between H. marismortui and E. coli, those that are notconserved between H. marismortui and rat, those that are not conservedbetween E. coli and rat, and those that are not conserved betweeneubacteria and eukaryota. The non conserved residues were identified asdescribed previously with respect to Table 5. TABLE 8 Residues thatDefine the Ribosomal Peptide Exit Tunnel Residues Not Residues NotResidues Not Conserved between Conserved Between Conserved BetweenResidue Conserved H. marismortui H. marismortui H. marismortui E. Coliand Rattus Between Bacteria A-site Residues and E. coli and Rattus(Coli/Rat) and Eukaryotes? 23S rRNA G23 Yes G24 Yes A60 Yes G88 Yes G89Yes A90 Yes U454 Yes A462 C C/A No A466 Yes G467 C A C/A No U468 C C YesG469 Yes A476 Yes A477 Yes C478 A A Yes G487 Yes . U488 A A Yes A489 YesC490 Yes C491 U C/U No C492 U C/U No A497 Yes A498 G A/G No G499 A G/ANo G500 G501 A G/A No A513 G A/G No G514 A A/G No G636 A G/A No A767 YesU768 A A Yes U835 del del/U No C839 U G U/G No U840 Yes A841 G G YesA844 Yes U845 A A Yes C879 Yes A882 Yes U883 Yes C884 Yes G885 A C A/CNo U888 C C Yes C889 Yes C890 G A G/A No U1359 Yes C1360 G G/C No C1361U C/U No U1362 Yes G1363 Yes G1364 A A/G No C1365 U C/U No C1366 A G A/GNo A1367 U G U/G No A1424 U G U/G No G1425 C U C/U No C1426 G C/G NoA1427 Yes C1428 A A Yes U1429 C C Yes G1430 A G/A No C1439 G A G/A NoU1440 G C G/C No G1441 C A C/A No A1442 U A/U No A1689 C A/C No C1690 AC/A No A1691 G A/G No A1836 Yes G1837 U A U/A No U1838 Yes A2054 YesA2055 Yes C2056 A A Yes U2057 C U/C No G2058 U C U/C No C2098 G A G/A NoG2099 A A/G No A2100 Yes A2101 Yes G2102 Yes A2103 Yes C2104 Yes C2105Yes A2474 Yes C2475 Yes C2476 U U/C No C2477 A C/A No U2478 C C YesG2482 Yes A2485 Yes A2486 Yes C2487 Yes A2488 U A/U No C2536 Yes A2538Yes G2540 Yes U2541 Yes C2542 Yes C2608 Yes G2611 Yes G2616 Yes G2618Yes U2619 Yes U2620 Yes U2621 Yes A2637 Yes G2643 Yes C2644 U U YesU2645 C C/U No G2646 C U C/U No C2647 U C/U No Protein L4 E59 Yes S60 VS V/S No F61 T W T/W No G62 Yes S63 T S/T No G64 Yes R65 K K/R No G66DEL A DEL/A No Q67 DEL V DEL/V No A68 DEL DEL/A No H69 K R K/R No V70 PI P/I No P71 W P W/P No K72 R R Yes L73 K R K/R No D74 G G Yes G75 T GT/G No R76 G G Yes A77 R T R/T No Protein L22 E20 H N H/N No E121 S KS/K No Q122 M M Yes Q123 K R K/R No G124 R R Yes R125 I R I/R No K126 MT M/T No P127 Y P/Y No R128 Yes A129 Yes M130 K H K/H No G131 Yes R132Yes A133 I A/I No S134 D N D/N No A135 R P R/P No W136 I Y I/Y No N137 LM L/M No Q140 T P T/P No Protein L39E ** N18 S19 R20 V21 P22 A23 Y24 V25M26 L27 K28 T29 E31 R35 N36 H37 K38 R39 R40 H41 R44 N45**Given that homologues for the H. marismortui ribosomal protein L39Eare not currently known, sequence comparisons with E. coli, and Rattusnorvegicus are not possible.Residues were determined by a 10 angstrom distance measurement betweenthe atoms of a model of a newly synthesized peptide positioned in thecenter of the exit tunnel and the 50S ribosome using the programSPOCK^(a). Conserved residues were determined by comparison between theproposed secondary structures of H. marismortui ^(b), E. coli ^(b), andRattus norvegicus ^(c).^(a)Christopher, Jon A. (1998) SPOCK: The Structural PropertiesObservation and Calculation Kit (Program Manual), The Center forMacromolecular Design, Texas A&M University, College Station, TX.^(b)Comparative RNA Web Site URL: http://www.rna.icmb.utexas.edu/c) Wool, I. G. (1985). “Studies of the Structure of Eukaryotic(Mammalian) Ribosomes” in Structure, Function, and Genetics ofRibosomes, supra, pp. 391-411.

FIG. 26 shows a region of the large ribosomal subunit in which anantibiotic binds. FIG. 26(A) shows an enlarged portion of the largeribosomal subunit with the antibiotic tylosin bound at the top of thepolypeptide exit tunnel adjacent the peptidyl transferase site. FIGS.26(B) and 26(C) are views showing each half of a large ribosomal subunitcut along the polypeptide exit tunnel and are provided to orient thereader to show the tylosin binding site relative to the large ribosomalunit as a whole. FIG. 26(A) also shows two cavities defined by the wallof the polypeptide exit tunnel and are denoted as “cavity 1” and “cavity2.” In addition, FIG. 26(A) also shows a disaccharide binding pocket.The direction in which the newly synthesized polypeptide chains exitsthe ribosome through the polypeptide exit tunnel is denoted by an arrow.

Table 9 identifies the residues in the H. marismortui 50S ribosomalsubunit that together define a first cavity within the wall ofpolypeptide exit tunnel (cavity 1). In addition, Table 9 identifieswhich of those residues that define cavity 1 are not conserved betweenH. marismortui and E. coli, those that are not conserved between H.marismortui and rat, those that are not conserved between E. coli andrat, and those that are not conserved between eubacteria and eukaryota.The non-conserved residues were identified as described previously withrespect to Table 5. TABLE 9 Residues that Define Cavity 1 in theRibosomal Peptide Exit Tunnel Residues Not Residues Not Residues NotConserved between Conserved Between Conserved Between Residue ConservedH. marismortui H. marismortui H. marismortui E. Coli and Rattus BetweenBacteria Residues and E. coli and Rattus (Coli/Rat) and Eukaryotes? C474G G Yes A766 Yes A767 Yes U768 A C A/C No U883 Yes C884 Yes G885 A A/GNo A886 Yes U888 C C Yes C889 Yes C890 G A G/A No U1359 Yes G1837 U AU/A No A2100 Yes A2101 Yes G2102 Yes A2103 Yes C2475 Yes C2476 U U/C NoC2477 A C/A No U2478 C C Yes A2479 G G/A No A2538 Yes Protein L4 P57 R SR/S No A58 Yes E59 Yes S60 V V/S No F61 T W T/W No G62 Yes S63 T S/T NoG64 Yes R65 K K/R No Q67 DEL V DEL/V No V70 P I P/I No P71 W W/P No K72R R Yes L73 K R K/R No D74 G G Yes G75 T T/G No R76 G G YesCavity residues were identified using the program SPOCK^(a). Conservedresidues were determined by comparison between the proposed secondarystructures of H. marismortui ^(b), E. coli ^(b), and Rattus norvegicus^(c).^(a)Christopher, Jon A. (1998) SPOCK: The Structural PropertiesObservation and Calculation Kit (Program Manual), The Center forMacromolecular Design, Texas A&M University, College Station, TX.^(b)Comparative RNA Web Site URL: http://www.rna.icmb.utexas.edu/^(c)Wool, I. G. (1985). “Studies of the Structure of Eukaryotic(Mammalian) Ribosomes” in Structure, Function, and Genetics ofRibosomes, supra, pp. 391-411.

Table 10 identifies the residues in the H. marismortui SOS ribosomalsubunit that together define a second cavity in the wall of polypeptideexit tunnel (cavity 2). In addition, Table 10 identifies which of thoseresidues that define cavity 2 are not conserved between H. marismortuiand E. coli, those that are not conserved between H. marismortui andrat, those that are not conserved between E. coli and rat, and thosethat are not conserved between eubacteria and eukaryota. The nonconserved residues were identified as described previously with respectto Table 5. TABLE 10 Residues that Define Cavity 2 in the RibosomalPeptide Exit Tunnel Residues Not Residues Not Residues Not Conservedbetween Conserved Between Conserved Between Residue Conserved H.marismortui H. marismortui H. marismortui E. Coli and Rattus BetweenBacteria Residues and E. coli and Rattus (Coli/Rat) and Eukaryotes? U831C G C/G No U832 DEL DEL/U No G833 U C U/C No G834 A C A/C No U835 DELDEL/U No G836 A A/G No U837 A U/A No C838 G G/C No C839 U G U/G No U840Yes A841 G G Yes A843 Yes A844 Yes U845 A A Yes A846 U A/U No C847 U U/CNo C848 U G U/G No C849 A G A/G No C1753 Yes A1754 Yes G1837 U A U/A NoU1838 Yes A1839 Yes G2099 A A/G No A2100 Yes A2103 Yes U2615 Yes G2616Yes U2621 Yes A2622 Yes G2643 Yes C2644 U U Yes U2645 C C/U No G2646 C UC/U No C2647 U C/U NoCavity residues were determined using the program SPOCK^(a). Conservedresidues were determined by comparison between the proposed secondarystructures of H. marismortui ^(b), E. coli ^(b), and Rattus norvegicus^(c).^(a)Christopher, Jon A. (1998) SPOCK: The Structural PropertiesObservation and Calculation Kit (Program Manual), The Center forMacromolecular Design, Texas A&M University, College Station, TX.^(b)Comparative RNA Web Site URL: http://www.rna.icmb.utexas.edu/^(c)Wool, I. G. (1985). “Studies of the Structure of Eukaryotic(Mammalian) Ribosomes” in Structure, Function, and Genetics ofRibosomes, supra, pp. 391-411.

Tables 9 and 10, however, define only two or many cavities disposedwithin the wall of the polypeptide exit tunnel. However, by using theatomic co-ordinates and molecular modeling methodologies describedherein, the skilled artisan may identify the residues (contributed byamino acids, nucleotides or a combination of both) that together defineother cavities within the wall of the polypeptide exit tunnel.

In addition, by using the atomic co-ordinates described herein, theskilled artisan can identify the antibiotic binding site of anyantibiotic of interest. This information also provides contact sitesbetween an antibiotic and the residues in a ribosome or ribosomalsubunit, which can be used to advantage in the design of novel ormodified protein synthesis inhibitors. The binding or contact sites fora variety of antibiotics are discussed in more detail below.

Table 11 identifies the residues in the H. marismortui 50S ribosomalsubunit that together define at least a portion of an anisomycin bindingpocket. In addition, Table 11 identifies which of those residues thatdefine at least a portion of the anisomycin binding pocket are notconserved between H. marismortui and E. coli, those that are notconserved between H. marismortui and rat, those that are not conservedbetween E. coli and rat, and those that are not conserved betweeneubacteria and eukaryota. The non-conserved residues were identified asdescribed previously with respect to Table 5. TABLE 11 Residues thatDefine the Anisomycin Binding Pocket Residues Not Residues Not ResiduesNot Conserved between Conserved Between Conserved Between ResidueConserved H. marismortui H. marismortui H. marismortui E. Coli andRattus Between Bacteria Residues and E. coli and Rattus (Coli/Rat) andEukaryotes? G2102 Yes G2482 Yes A2486 Yes C2487 Yes A2488 U A/U No U2535Yes A2538 Yes U2539 Yes G2540 Yes U2541 YesResidues were determined by a 5.8 angstrom distance measurement betweenthe atoms of anisomysin and the 50S ribosome using the programSPOCK^(a). Conserved residues were determined by comparison between theproposed secondary structures of H. marismortui ^(b), E. coli ^(b), andRattus norvegicus ^(c).^(a)Christopher, Jon A. (1998) SPOCK: The Structural PropertiesObservation and Calculation Kit (Program Manual), The Center forMacromolecular Design, Texas A&M University, College Station, TX.^(b)Comparative RNA Web Site URL: http://www.rna.icmb.utexas.edu/^(c)Wool, I. G. (1985). “Studies of the Structure of Eukaryotic(Mammalian) Ribosomes” in Structure, Function, and Genetics ofRibosomes, supra, pp. 391-411.

Table 12 identifies the residues in the H. marismortui 50S ribosomalsubunit that together define at least a portion of a blasticidin bindingpocket. As demonstrated in Table 12, all of the residues in that portionof the blasticidin binding pocket are conserved between H. marismortuiand E. coli, between H. marismortui and rat, between E. coli and rat,and between eubacteria and eukaryota, as determined using the comparisonmethod described previously with respect to Table 5. TABLE 12 Residuesthat Define the Blasticidin Binding Pocket Residues Not Residues NotResidues Not Conserved between Conserved Between Conserved BetweenResidue Conserved H. marismortui H. marismortui H. marismortui E. Coliand Rattus Between Bacteria Residues and E. coli and Rattus (Coli/Rat)and Eukaryotes? C2104 Yes C2105 Yes C2106 Yes G2284 Yes G2285 Yes U2473Yes A2474 Yes A2485 Yes A2486 Yes U2620 Yes G2634 Yes A2635 Yes C2636Yes A2637 YesResidues were determined by a 5.8 angstrom distance measurement betweenthe atoms of Blasticidin and the 50S ribosome using the programSPOCK^(a). Conserved residues were determined by comparison between theproposed secondary structures of H. marismortui ^(b), E. coli ^(b), andRattus norvegicus ^(c).^(a)Christopher, Jon A. (1998) SPOCK: The Structural PropertiesObservation and Calculation Kit (Program Manual), The Center forMacromolecular Design, Texas A&M University, College Station, TX.^(b)Comparative RNA Web Site URL: http://www.rna.icmb.utexas.edu/^(c)Wool, I. G. (1985). “Studies of the Structure of Eukaryotic(Mammalian) Ribosomes” in Structure, Function, and Genetics ofRibosomes, supra, pp. 391-411.

Table 13 identifies the residues in the H. marismortui 50S ribosomalsubunit that together define at least a portion of a carbomycin bindingpocket. In addition, Table 13 identifies which of those residues thatdefine at least a portion of the carbomycin binding pocket are notconserved between H. marismortui and E. coli, those that are notconserved between H. marismortui and rat, those that are not conservedbetween E. coli and rat, and those that are not conserved betweeneubacteria and eukaryota. The non-conserved residues were identified asdescribed previously with respect to Table 5. TABLE 13 Residues thatDefine the Carbomvcin Binding Pocket Residues Not Residues Not ResiduesNot Conserved between Conserved Between Conserved Between ResidueConserved H. marismortui H. marismortui H. marismortui E. Coli andRattus Between Bacteria Residues and E. coli and Rattus (Coli/Rat) andEukaryotes? C839 U G U/G No G2099 A A/G No A2100 Yes G2102 Yes A2103 YesC2104 Yes A2486 Yes C2487 Yes A2538 Yes G2540 Yes U2541 Yes U2620 YesC2644 U U Yes G2646 C U C/U NoResidues were determined by a 5.8 angstrom distance measurement betweenthe atoms of carbomycin and the 50S ribosome using the programSPOCK^(a).Conserved residues were determined by comparison between the proposedsecondary structures of H. marismortui ^(b), E. coli ^(b), and Rattusnorvegicus ^(c).^(a)Christopher, Jon A. (1998) SPOCK: The Structural PropertiesObservation and Calculation Kit (Program Manual), The Center forMacromolecular Design, Texas A&M University, College Station, TX.^(b)Comparative RNA Web Site URL: http://www.rna.icmb.utexas.edu/^(c)Wool, I. G. (1985). “Studies of the Structure of Eukaryotic(Mammalian) Ribosomes” in Structure, Function, and Genetics ofRibosomes, supra, pp. 391-411.

Table 14 identifies the residues in the H. marismortui 50S ribosomalsubunit that together define at least a portion of a tylosin bindingpocket. In addition, Table 14 identifies which of those residues thatdefine at least a portion of the tylosin binding pocket are notconserved between H. marismortui and E. colii, those that are notconserved between H. marismortui and rat, those that are not conservedbetween E. colii and rat, and those that are not conserved betweeneubacteria and eukaryota. The non-conserved residues were identified asdescribed previously with respect to Table 5. TABLE 14 Residues thatDefine the Tylosin Binding Pocket Residues Not Residues Not Residues NotConserved between Conserved Between Conserved Between Residue ConservedH. marismortui H. marismortui H. marismortui E. Coli and Rattus BetweenBacteria Residues and E. coli and Rattus (Coli/Rat) and Eukaryotes? C839U G U/G No A841 G G Yes A843 Yes A844 Yes U845 A A Yes G1837 U A U/A NoC2098 G A G/A No G2099 A A/G No A2100 Yes G2102 Yes A2103 Yes A2538 YesU2539 Yes G2540 Yes U2541 Yes U2645 C C/U No G2646 C U C/U NoResidues were determined by a 5.8 angstrom distance measurement betweenthe atoms of the tylosin and the 50S ribosome using the the programSPOCK^(a). Conserved residues were determined by comparison between theproposed secondary structures of H. marismortui ^(b), E. coli ^(b), andRattus norvegicus ^(c).^(a)Christopher, Jon A. (1998) SPOCK: The Structural PropertiesObservation and Calculation Kit (Program Manual), The Center forMacromolecular Design, Texas A&M University, College Station, TX.^(b)Comparative RNA Web Site URL: http://www.rna.icmb.utexas.edu/^(c)Wool, I. G. (1985). “Studies of the Structure of Eukaryotic(Mammalian) Ribosomes” in Structure, Function, and Genetics ofRibosomes, supra, pp. 391-411.

Table 15 identifies the residues in the H. marismortui 50S ribosomalsubunit that together define at least a portion of a sparsomycin bindingpocket. As demonstrated in Table 15, all of the residues in that portionof the sparsomycin binding pocket are conserved between H. marismortuiand E. coli, between H. marismortui and rat, between E. coli and rat,and between eubacteria and eukaryota, as determined using the comparisonmethod described previously with respect to Table 5. TABLE 15 Residuesthat Define the Sparsomycin Binding Pocket Residues Not Residues NotResidues Not Conserved between Conserved Between Conserved BetweenResidue Conserved H. marismortui H. marismortui H. marismortui E. Coliand Rattus Between Bacteria Residues and E. coli and Rattus (Coli/Rat)and Eukaryotes? A2486 Yes C2487 Yes U2541 Yes C2608 Yes U2619 Yes U2620Yes C2636 Yes A2637 YesResidues were determined by a 5.8 angstrom distance measurement betweenthe atoms of sparsomycin and the 50S ribosome using the the programSPOCK^(a). Conserved residues were determined by comparison between theproposed secondary structures of H. marismortui ^(b), E. coli ^(b), andRattus norvegicus ^(c).^(a)Christopher, Jon A. (1998) SPOCK: The Structural PropertiesObservation and Calculation Kit (Program Manual), The Center forMacromolecular Design, Texas A&M University, College Station, TX.^(b)Comparative RNA Web Site URL: http://www.rna.icmb.utexas.edu/^(c)Wool, I. G. (1985). “Studies of the Structure of Eukaryotic(Mammalian) Ribosomes” in Structure, Function, and Genetics ofRibosomes, supra, pp. 391-411.

Table 16 identifies the residues in the H. marismortui 50S ribosomalsubunit that together define at least a portion of a virginiamycinbinding pocket. As demonstrated in Table 16, all of the residues in thatportion of the virginiamycin binding pocket are conserved between H.marismortui and E. coli, between H. marismortui and rat, between E. coliand rat, and between eubacteria and eukaryota, as determined using thecomparison method described previously with respect to Table 5. TABLE 16Residues that Define the Virginiamycin Binding Pocket Residues NotResidues Not Residues Not Conserved between Conserved Between ConservedBetween Residue Conserved H. marismortui H. marismortui H. marismortuiE. Coli and Rattus Between Bacteria Residues and E. coli and Rattus(Coli/Rat) and Eukaryotes? A2100 Yes G2102 Yes A2103 Yes C2104 Yes C2105Yes G2482 Yes A2486 Yes C2487 Yes U2535 Yes C2536 Yes A2538 Yes U2539Yes G2540 Yes U2541 Yes U2620 YesResidues were determined by a 5.8 angstrom distance measurement betweenthe atoms of virginiamycin and the 50S ribosome using the the programSPOCK^(a). Conserved residues were determined by comparison between theproposed secondary structures of H. marismortui ^(b), E. coli ^(b), andRattus norvegicus ^(c).^(a)Christopher, Jon A. (1998) SPOCK: The Structural PropertiesObservation and Calculation Kit (Program Manual), The Center forMacromolecular Design, Texas A&M University, College Station, TX.^(b)Comparative RNA Web Site URL: http://www.rna.icmb.utexas.edu/^(c)Wool, I. G. (1985). “Studies of the Structure of Eukaryotic(Mammalian) Ribosomes” in Structure, Function, and Genetics ofRibosomes, supra, pp. 391-411.

Table 17 identifies the residues in the H. marismortui 50S ribosomalsubunit that together define at least a portion of a spiramycin bindingpocket. In addition, Table 17 identifies which of those residues thatdefine at least a portion of the spiramycin binding pocket are notconserved between H. marismortui and E. coli, those that are notconserved between H. marismortui and rat, those that are not conservedbetween E. coli and rat and those that are not conserved betweeneubacteria and eukaryota. The non-conserved residues were identified asdescribed previously with respect to Table 5. TABLE 17 Residues thatDefine the Spiramycin Binding Pocket Residues Not Residues Not ResiduesNot Conserved between Conserved Between Conserved Between ResidueConserved H. marismortui H. marismortui H. marismortui E. Coli andRattus Between Bacteria Residues and E. coli and Rattus (Coli/Rat) andEukaryotes? C839 U G U/G No C2098 G A G/A No G2099 A A/G No A2100 YesG2102 Yes A2103 Yes A2538 Yes U2539 Yes G2540 Yes U2541 Yes C2644 U UYes G2646 C U C/U NoResidues were determined by a 5.8 angstrom distance measurement betweenthe atoms of spiramycin and the 50S ribosome using the the programSPOCK^(a). Conserved residues were determined by comparison between theproposed secondary structures of H. marismortui ^(b), E. coli ^(b), andRattus norvegicus ^(c).^(a)Christopher, Jon A. (1998) SPOCK: The Structural PropertiesObservation and Calculation Kit (Program Manual), The Center forMacromolecular Design, Texas A&M University, College Station, TX.^(b)Comparative RNA Web Site URL: http://www.rna.icmb.utexas.edu/^(c)Wool, I. G. (1985). “Studies of the Structure of Eukaryotic(Mammalian) Ribosomes” in Structure, Function, and Genetics ofRibosomes, supra, pp. 391-411.

The skilled artisan, when in possession of the foregoing or otherexemplary target sites, may use the process of rational drug design toidentify molecules that potentially bind to one or more of the targetsites and/or inhibit ribosomal activity. Furthermore, by taking intoaccount which of the residues that define the target site are conservedbetween pathogens but not conserved between host species, the skilledartisan can design new species-specific protein synthesis inhibitors. Itis apparent that the skilled artisan can take advantage of the regionsthat are not conserved between E. coli and rat to provide target regionsfor rational drug design. By way of example, FIG. 29 shows certainregions of the polypeptide exit tunnel that are conserved between E.coli and rat (denoted in red) and regions of the polypeptide exit tunnelthat are not conserved between E. coli and rat (denoted in blue). FIGS.29(A) and 29(B) provide enlarged views of a large ribosomal subunit whencut in half along the polypeptide exit tunnel. FIG. 29(C) is provided toorient the reader to the view in FIG. 29(A) relative to the largeribosomal subunit. FIG. 29(D) is provided to orient the reader to theview in FIG. 29B relative to the large ribosomal subunit. In addition,the skilled artisan when in possession of mutations that prevent orreduce antibiotic activity (i.e., are related to antibiotic resistance)can use this information to model the relevant antibiotic bindingproduct which can then be used as a basis for rational drug design toidentify small molecules that overcome drug resistance. It iscontemplated that a variety of computer modeling procedures, forexample, homology modeling protocols, can be used to provide a model ofa drug resistance target site by implementing site directed mutagenesisof nucleotides and/or amino acids and then using the appropriate energyminimization and refinement protocols.

c. Identification of Candidate Molecules.

It is contemplated that candidate molecules that inhibit proteinbiosynthesis can be designed entirely de novo or may be based upon apre-existing protein biosynthesis inhibitor. Either of these approachescan be facilitated by computationally screening databases and librariesof small molecules for chemical entities, agents, ligands, or compoundsthat can bind in whole, or in part, to ribosomes and ribosomal subunits,more preferably to large ribosomal subunits, and even more preferably to50S ribosomal subunits. In this screening, the quality of fit of suchentities or compounds to the binding site or sites may be judged eitherby shape complementarity or by estimated interaction energy (Meng et al(1992) J. Coma. Chem. 13: 505-524).

The design of molecules that bind to or inhibit the functional activityof ribosomes or ribosomal subunits according to this invention generallyinvolves consideration of two factors. First, the molecule must becapable of physically and structurally associating with the largeribosomal subunit. Non-covalent molecular interactions important in theassociation of ribosomes and ribosomal subunits with the molecule,include hydrogen bonding, van der Waals and hydrophobic interactions.Second, the molecule must be able to assume a conformation that allowsit to associate with the ribosomes or ribosomal subunits, morepreferably with the large ribosomal subunits, and even more preferablywith the 50S ribosomal subunit. Although certain portions of themolecule may not directly participate in this association with aribosome or ribosomal subunits those portions may still influence theoverall conformation of the molecule. This, in turn, may have asignificant impact on binding affinities, therapeutic efficacy,drug-like qualities, and potency. Such conformational requirementsinclude the overall three-dimensional structure and orientation of thechemical entity or molecule in relation to all or a portion of theactive site or other region of the ribosomes or ribosomal subunits, orthe spacing between functional groups of a molecule comprising severalchemical entities that directly interact with the ribosomes or ribosomalsubunits, more preferably with the large ribosomal subunits, and evenmore preferably with the 50S ribosomal subunit.

The potential, predicted, inhibitory or binding effect of a molecule onribosomes and ribosomal subunits may be analyzed prior to its actualsynthesis and testing by the use of computer modeling techniques. If thetheoretical structure of the given molecule suggests insufficientinteraction and association between it and ribosomes or ribosomalsubunits, synthesis and testing of the molecule is obviated. However, ifcomputer modeling indicates a strong interaction, the molecule may thenbe synthesized and tested for its ability to interact with the ribosomesor ribosomal subunits and inhibit protein synthesis. In this manner,synthesis of inoperative molecules may be avoided. In some cases,inactive molecules are synthesized predicted on modeling and then testedto develop a SAR (structure-activity relationship) for moleculesinteracting with a specific region of the ribosome or ribosomal subunit,more preferably of the large ribosomal subunit, and even more preferablyof the 50S ribosomal subunit. As used herein, the term “SAR”, shallcollectively refer to the structure-activity/structure propertyrelationships pertaining to the relationship(s) between a compound'sactivity/properties and its chemical structure.

d. De Novo Design.

One skilled in the art may use one of several methods to identifychemical moieties or entities, compounds, or other agents for theirability to associate with a preselected target site within a ribosomesor ribosomal subunit. This process may begin by visual inspection orcomputer assisted modeling of, for example, the target site on thecomputer screen based on the atomic co-ordinates of the 50S ribosomalsubunit and/or its complexes with other analogues and antibiotics,deposited in the RCSB Protein Data Bank with accession numbers PDB ID:1FFK, 1JJ2, 1FFZ, or 1FG0, and/or listed in a table contained on DiskNo. 1, 2 or 3 of 3. In one embodiment, compound design uses computermodeling programs which calculate how different molecules interact withthe various sites of the ribosome, ribosomal subunit, or a fragmentthereof. Selected chemical moieties or entities, compounds, or agentsmay then be positioned in a variety of orientations, or docked, withinat least a portion of the target site of a ribosome or ribosomalsubunit, more preferably of a large ribosomal subunit, and even morepreferably of a 50S ribosomal subunit. Databases of chemical structuresare available from, for example, Cambridge Crystallographic Data Center(Cambridge, U.K.) and Chemical Abstracts Service (Columbus, Ohio).Docking may be accomplished using software such as Quanta and Sybyl,followed by energy minimization and molecular dynamics with standardmolecular mechanics forcefields, such as CHARMM and AMBER.

Specialized computer programs may also assist in the process ofselecting chemical entities. These include, but are not limited to:

(1) GRID (Goodford, P. J., “A Computational Procedure for DeterminingEnergetically Favorable Binding Sites on Biologically ImportantMacromolecules” (1985) J. Med. Chem. 28, 849-857). Software such asGRID, a program that determines probable interaction sites betweenprobes with various functional group characteristics and themacromolecular surface, can be used to analyze the surface sites todetermine structures of similar inhibiting proteins or molecules. TheGRID calculations, with suitable inhibiting groups on molecules (e.g.,protonated primary amines) as the probe, are used to identify potentialhotspots around accessible positions at suitable energy contour levels.GRID is available from Oxford University, Oxford, UK.

(2) MCSS (Miranker, A. and M. Karplus (1991) “Functionality Maps ofBinding Sites: A Multiple Copy Simultaneous Search Method.” Proteins:Structure, Function and Genetics 11: 29-34). MCSS is available fromMolecular Simulations, Burlington, Mass.

(3) AUTODOCK (Goodsell, D. S. and A. J. Olsen (1990) “Automated Dockingof Substrates to Proteins by Simulated Annealing” Proteins: Structure,Function, and Genetics 8: 195-202). AUTODOCK is available from ScrippsResearch Institute, La Jolla, Calif.

(4) DOCK (Kuntz, I. D. et al. (1982) “A Geometric Approach toMacromolecule-Ligand Interactions” J. Mol. Biol. 161: 269-288). Theprogram DOCK may be used to analyze an active site or ligand bindingsite and suggest ligands with complementary steric properties. DOCK isavailable from University of California, San Francisco, Calif.

(5) ALADDIN (Van Drie et al. (1989) “ALADDIN: An Integrated Tool ofComputer Assisted Molecular Design and Pharmacophore Recognition FromGeometric, Steric and Substructure Searching of Three-DimensionalStructures” J. Comp-Aided Mol. Des. 3: 225).

(6) CLIX (Davie and Lawrence (1992) “CLIX: A Search Algorithm forFunding Novel Ligands Capable of Binding Proteins of KnownThree-Dimensional Structure” Proteins 12: 31-41).

(7) GROUPBUILD (Rotstein and Murcko (1993) “GroupBuild: A Fragment-BasedMethod for De Novo Drug Design” J. Med Chem 36: 1700).

(8) GROW (Moon and Howe (1991) “Computer Design of Bioactive Molecules:A Method for Receptor-Based De Novo Ligand Design” Proteins 11: 314).

Once suitable chemical moieties or entities, compounds, or agents havebeen selected, they can be assembled into a single molecule. Assemblymay proceed by visual inspection and/or computer modeling andcomputational analysis of the spatial relationship of the chemicalmoieties or entities, compounds or agents with respect to one another inthree-dimensional space. This could then be followed by model buildingusing software such as Quanta or Sybyl.

Useful programs to aid one of skill in the art in connecting theindividual chemical entities, compounds, or agents include but are notlimited to:

(1) CAVEAT (Bartlett, P. A. et al. (1989) “CAVEAT: A Program toFacilitate the Structure-Derived Design of Biologically ActiveMolecules”. In molecular Recognition in Chemical and BiologicalProblems”, Special Pub., Royal Chem. Soc. 78: 82-196) and (Bacon et al.(1992) J. Mol. Biol. 225: 849-858). CAVEAT uses databases of cycliccompounds which can act as “spacers” to connect any number of chemicalfragments already positioned in the active site. This allows one skilledin the art to quickly generate hundreds of possible ways to connect thefragments already known or suspected to be necessary for tight binding.CAVEAT is available from the University of California, Berkeley, Calif.

(2) 3D Database systems such as MACCS-3D (MDL Information Systems, SanLeandro, (CA). This area is reviewed in Martin, Y. C., (1992) “3DDatabase Searching in Drug Design”, J. Med. Chem. 35: 2145-2154.

(3) HOOK (available from Molecular Simulations, Burlington, Mass.).

Instead of proceeding to build a molecule of interest in a step-wisefashion one chemical entity at a time as described above, the moleculeof interest may be designed as a whole using either an empty active siteor optionally including some portion or portions of a known inhibitor orinhibitors. Software that implements these methods include:

(1) LUDI (Bohm, H.-J. (1992) “The Computer Program LUDI: A New Methodfor the De Novo Design of Enzyme Inhibitors”, J. ComR. Aid. Molec.Design 6: 61-78). The program LUDI can determine a list of interactionsites into which to place both hydrogen bonding and hydrophobicfragments. LUDI then uses a library of approximately 600 linkers toconnect up to four different interaction sites into fragments. Thensmaller “bridging” groups such as —CH₂— and —COO— are used to connectthese fragments. For example, for the enzyme DHFR, the placements of keyfunctional groups in the well-known inhibitor methotrexate werereproduced by LUDI. See also, Rotstein and Murcko, (1992) J. Med. Chem.36:1700-1710. LUDI is available from Biosym Technologies, San Diego,Calif.

(2) LEGEND (Nishibata, Y. and A. Itai (1991) Tetrahedron 47, 8985).LEGEND is available from Molecular Simulations, Burlington, Mass.

(3) LeapFrog (available from Tripos Associates, St. Louis, Mo.).

(4) Aladdin (available from Daylight Chemical Information Systems,Irvine, Calif.)

Other molecular modeling techniques may also be employed in accordancewith this invention. See, e.g., Cohen, N. C. et al. (1990) “MolecularModeling Software and Methods for Medicinal Chemistry, J. Med. Chem. 33:883-894. See also, Navia, M. A. and M. A. Murcko (1992) “The Use ofStructural Information in Drug Design”, Current Opinions in StructuralBiology 2: 202-210; and Jorgensen (1998) “BOSS—Biochemical and OrganicSimulation System” in the Encyclopedia of Computational Chemistry (P. V.R. Schleyer, ed.) Wiley & Sonstra., Athens, U.S.A. 5: 3281-3285).

It is contemplated that during modeling, it may be possible to introduceinto the molecule of interest, chemical moieties that may be beneficialfor a molecule that is to be administered as a pharmaceutical. Forexample, it may be possible to introduce into or omit from the moleculeof interest, chemical moieties that may not directly affect binding ofthe molecule to the target area but which contribute, for example, tothe overall solubility of the molecule in a pharmaceutically acceptablecarrier, the bioavailability of the molecule and/or the toxicity of themolecule. Considerations and methods for optimizing the pharmacology ofthe molecules of interest can be found, for example, in “Goodman andGilman's The Pharmacological Basis of Therapeutics” Eighth Edition(Goodman Gilman, Rall, Nies, & Taylor (eds.)). Pergaman Press (1985);Jorgensen & Duffy (2000) Bioorg. Med. Chem. Lett. 10: 1155-1158.

Furthermore, the computer program “Qik Prop” can be used to providerapid predictions for physically significant descriptions andpharmaceutically-relevant properties of an organic molecule of interest.A ‘Rule of Five’ probability scheme can be used to estimate oralabsorption of the newly synthesized compounds (Lipinski et al. (1997)Adv. Drug Deliv. Rev. 23:3).

Programs suitable for pharmacophore selection and design include:

-   -   (1) DISCO (Abbot Laboratories, Abbot Park, Ill.).    -   (2) Catalyst (Bio-CAD Corp., Mountain View, Calif.).    -   (3) Chem DBS-3D (Chemical Design Ltd., Oxford, U.K.).

Furthermore, the skilled artisan may use the information available onhow to design suitable therapeutically active and pharmaceuticallyuseful compounds, and use this information in the design of new proteinsynthesis inhibitors of the invention. See, for example, Lipinski et al.(1997) Ad Drug Deliv. Reviews 23: 3-25; Van de Waterbeemd et al. (1996)Quantitative Structure-Activity Relationships 15: 480-490; and Crucianiet al (2000), Theochem-J. Mol. Struct. 503: 17-30.

The entry of the co-ordinates of the ribosome's or ribosomal subunit'sproteins and RNAs into the computer programs discussed above results inthe calculation of most probable structure of the macromolecule,including overall atomic co-ordinates of a ribosome, ribosomal subunitor a fragment thereof. These structures can be combined and refined byadditional calculations using such programs to determine the probable oractual three-dimensional structure of the ribosome, ribosomal subunit ora fragment thereof, including potential or actual active or bindingsites of ligands.

e. Modification of Existing Molecules.

Instead of designing molecules of interest entirely de novo it iscontemplated that pre-existing molecules or proteins thereof may be usedas a starting point for the design of a new candidate. It iscontemplated that many of the approaches useful for designing moleculesde novo may also be useful for modifying existing molecules.

It is contemplated that knowledge of the spatial relationship between aprotein biosynthesis inhibitor, for example, an antibiotic, and itsrespective binding site within a ribosome permits the design of modifiedinhibitors that may have better binding properties, for example, higherbinding affinity and/or specificity, relative to the molecule from whichit was derived. Alternatively, knowledge of inhibitor contact siteswithin a ribosome permits the synthesis of a new molecule that contain,for example, a portion of a first molecule that binds to the contactsite and another portion that contributes additional functionality.

It is contemplated that a variety of modified molecules (for example,modified antibiotics) may be designed using the atomic co-ordinatesprovided herein. For example, it is contemplated that by knowing thespatial relationship of one or more of antibiotics relative to the largeribosomal subunit it is possible to generate new antibiotic-basedmolecules. The atomic co-ordinates of each antibiotic relative to thelarge ribosomal subunit provides information on what portions of theribosome or ribosomal subunit and the antibiotic contact one another.Accordingly, from this information the skilled artisan may not onlyidentify contact locations within the ribosome that can be used for denovo drug design, as discussed above, but also may identify portions ofan antibiotic that can act as a ribosome binding domain.

Based on the information provided herein, the skilled artisan mayreadily identify and produce hybrid antibiotics that comprise a ribosomebinding domain of a first antibiotic and a ribosome binding domain of asecond, different antibiotic. The resulting hybrid antibioticspreferably bind to each of respective contact locations within theribosomal subunit simultaneously. The atomic co-ordinates providedherein permit the skilled artisan to identify candidate antibiotics thatmay be used as templates in the synthesis of a hybrid, and also providesteric information necessary to produce linking chemistries such thateach ribosome binding domain is properly orientated relative to itsrespective contact site. As a result, it is contemplated that theskilled artisan may produce a hybrid antibiotic that binds to a ribosomeor ribosomal subunit with a higher affinity and/or have higher proteinsynthesis inhibitory activity than either of the individual templateantibiotics used to generate the hybrid. Alternatively, the hybridantibiotic may overcome resistance phenotypes that may have developedagainst either of the template antibiotics. For example, the proximityof the site occupied by the disaccharide moiety of carbomycin to thesite filled by anisomycin suggests that a hybrid compound includingportions of both carbomycin and anisomycin may be an effective inhibitorof protein synthesis.

Furthermore, the atomic co-ordinates provided herein permit the skilledartisan to use the information pertaining to identify a ribosome bindingdomain and to design other types of protein synthesis inhibitors. Forexample, with an understanding of the ribosome contact region and thesurrounding environment, the skilled artisan can provide novelmolecules, a portion of which is based upon the antibiotic bindingregion (binding domain) and another portion of which (effector domain)can be designed as a novel space filling domain that sterically inhibitsor disrupts protein biosynthesis within the ribosome or secretionthrough the polypeptide exit tunnel. For example, the skilled artisanmay combine the ribosome binding region of the antibiotic, tylosin,which binds to one side of the polypeptide exit tunnel close to thepeptidyl transferase site, with a novel chemical moiety bulky enough toblock the polypeptide exit tunnel. However, it is contemplated that theskilled artisan may take advantage of one or more of the many of theantibiotic contact regions disclosed herein to design entirely newbinding and effector domains.

Furthermore, the present invention permits the skilled artisan to designmolecules, for example, selective protein synthesis inhibitors that aretailored to be more potent with respect to ribosomes of a targetorganism, for example, a pathogen such a microbe, and less potent, i.e.,less toxic, to ribosomes of a non target organism, for example, hostorganism such as a human. Also, the invention permits the skilledartisan to use the atomic co-ordinates and structures of the largeribosomal subunit and its complexes with protein synthesis inhibitors todesign modifications to starting compounds, such as an antibiotic, thatwill bind more tightly to a target ribosome (e.g., the 50S ribosomalsubunit of bacteria) and less tightly to a non-targeted ribosome (e.g.,human 60S ribosomal subunit).

The structure of a complex between the large ribosomal subunit and thestarting compound (e.g., tylosin or another protein synthesis inhibitor)can also be used to guide the modification of that compound to producenew compounds that have other desirable properties for the applicableindustrial and other uses (e.g., as pharmaceuticals, herbicides orinsecticides), such as chemical stability, solubility or membranepermeability.

A variety of antibiotics bind the large ribosomal subunit and disruptprotein synthesis and include members of antibiotic families whichinclude, for example, chloramphenicols, macrolides, lincosamides,streptogramins, althiomycins, oxazolinones, nucleotide analogs,thiostreptons, peptides, glutarimides, and trichothecenes.

Members of the chloramphenicol family include, for example,Chloramphenicol and Iodoamphenicol. Members of the macrolide familyinclude, for example, Biaxin (Clarithromycin), Zithromax(Azithromycins), Ketek (Telithromycin; ketolide), ABT-773, Tylosin,Spiramycin I, Spiramycin II, Spiramycin III, Erythromycin A, CarbomycinA, Telithromycin, Methymycin, Narbomycin, Lankamycin, Oleandomycin,Megalomycin, Chalcomycin, Niddamycin, Leucomycin, Angolamycin, andRelomycin. Members of the licosamide family include, for example,Clindamycin and Lincomycin. Members of the streptogramin family include,for example, Streptogramin A, Streptogramin B, Ostreogrycin G, Synercid,Virginiamycin S1, Virginiamycin S2, Virginiamycin S3, Virginiamycin S4,Vernamycin B, Vernamycin C, Patricin A, and Patricin B. A member of thealthiomycin family, includes, for example, Althiomycin. A member of theoxazolidine family, includes, for example, Linezolid. Members of thefamily of nucleotide analogs include, for example, Sparsomycin,Puromycin, Anisomycin, and Blasticidin S. Members of the thiostreptonfamily include, for example, Thiostrepton, Siomycin, Sporangiomycin, andThiopeptin. Members of the peptide family include, for example,Viomycin, Capreomycin IA, Capreomycin IB, Capreomycin IIA, andCapreomycin IIB. Members of the glutarimide family include, for example,Cycloheximide, Streptovitacins, Streptimidone, Inactone, Actiphenol.Members of the trichothecene family include, for example, Trichodermin,Trichodermol, Trichodermone, Vomitoxin, T-2 toxin, Trichothecin,Nivalenol, and Verrucarin A.

Inhibitors can be diffused into or soaked with the stabilized crystalsof the large ribosomal subunit as described in Example 3 to form acomplex with the large ribosomal subunit for collecting X-raydiffraction data. Alternatively, the inhibitors can be co-crystallizedwith the large ribosomal subunit by mixing the inhibitor with the largeribosomal subunit before precipitation with high salt.

Starting with the structure of the ribosome from H. marismortui, thestructure of the ribosome from a non-targeted organism (for example, thehuman 60S ribosomal subunit) can be constructed by homology modeling,i.e., by changing the structure of residues at a target site of interestfor the residues at the same positions in of the non-target ribosome.This is done computationally by removing the side chains from theribosome of known structure and replacing them by the side chains of theunknown structure put in sterically plausible positions. In this way, itcan be understood how the shapes of the target sites within the targetedand non-targeted ribosomes differ. This process, therefore, providesinformation concerning how a molecule that binds the target site can bechemically altered in order to produce molecules that will bind tightlyand specifically to the targeted ribosome but will simultaneously beprevented from binding to the non-targeted ribosome. Likewise, knowledgeof portions of the bound molecules that face the solvent permitintroduction of other functional groups for additional pharmaceuticalpurposes. The process of homology structure modeling can also be used tounderstand the mechanisms whereby mutant ribosomes become resistant tothe effects of pharmaceuticals or pesticides, such as herbicides orinsecticides. Furthermore, with knowledge of the portions of theribosomal subunit that participates in drug resistance, the skilledartisan may design new molecules that overcome the problem of drugresistance.

The use of homology structure modeling to design molecules that bindmore tightly to the target ribosome than to the non-target ribosome haswide-spread applicability. The methods outlined herein can be Used tocontrol any targeted organism, for example, a pathogen, by designingmolecules that inhibit large ribosomal subunits of the targetedorganisms while failing to inhibit the 50S or 60S ribosomal subunit ofthe non-targeted organism, for example, a host, to the same extent ornot at all. The molecules identified or prepared by the methods of thepresent invention can be used to control the targeted organisms whilecausing the non-targeted organism little or no adverse effects. Thus,the molecules identified or developed using the methods of the presentinvention can be designed so that their administration kills the targetorganisms or inhibits some aspect of the biological functions of thetarget organisms while failing to have a similar effect on thenon-targeted organism. The adverse effects of the agent on the targetedorganisms may include, but are not limited to, death of the targetorganism; slowing growth rates; slowing or eliminating passage from onegrowth phase to another (e.g., extending the larval growth stage);slowing or eliminating reproduction, decreasing or preventing mating,decreasing or eliminating offspring production, limiting or eliminatingtarget organism weight gains; decreasing or eliminating feeding abilityand behaviors; and disrupting cellular, tissue and/or organ functions.

The novel agents contemplated by the present invention can be useful asherbicides, pesticides (e.g., insecticides, nematocides, rodenticides,etc.), miticides, or antimicrobial agents (e.g., antifungals,antibacterials, antiprotozoals, etc.) to target specific organisms. Forexample, the novel agents can target animal and plant parasiticnematodes, prokaryotic organisms (disease causing microbes), andeukaryotic multicellular pests. Specific examples of multicellular pestsinclude, but are not limited to, insects, fungi, bacteria, nematodes,mites and ticks, protozoan pathogens, animal-parasitic liver flukes, andthe like.

Herbicides, pesticides miticides, and antimicrobial agents that inhibitprotein synthesis by interacting with ribosomes are known to the skilledartisan. A few examples are discussed below. These known agents can bemodified to obtain novel agents by using computer modeling techniquesand knowledge of the structure of ribosomes and ribosomal subunits andthe structure of ribosome/agent and ribosomal subunit/agent complexes.

The ketolide ABT-773 binds ribosomes tighter than erythromycin in S.pneumoniae and is able to defeat macrolide resistance in bacteria(Capobianco et al. (2000) Antimicrob Agents Chemother 44(6), 1562-1567).The tools and methodologies of the present invention can be used toobtain erythromycin derivatives that bind the ribosomes or ribosomalsubunits of target bacteria more tightly than they bind the ribosomesand ribosomal subunits of non-target animals. The target bacteria can beany infectious bacteria, particularly S. pneumoniae, and even moreparticularly erythromycin-resistant S. pneumoniae. The non-targetanimals can be any animal, particularly mammals, and even moreparticularly humans.

Examples of antibiotics that are inhibitors of protein synthesisinclude, but are not limited to, puromycin, cycloheximide,chloramphenicol, tetracycline, and streptomycin (Heldt, (1996) PlantBiochemistry and Molecular Biology 21.2: 458-464). Puromycin, asdiscussed earlier, binds as an analogue of an aminoacyl-tRNA to theA-site and is added to nascent peptide chains, its weak associate withthe ribosome prevents further elongation steps in prokaryotes andeukaryotes. Cycloheximide inhibits peptidyl transferase in eukaryoticribosomes. Chloramphenicol inhibits peptidyl transferase in prokaryoticribosomes. Tetracycline binds to the 30S subunit and inhibits thebinding of aminoacyl-tRNA to prokaryotic ribosomes much more than toeukaryotic ones. Streptomycin interacts with 30S ribosomes which resultsin an incorrect recognition of mRNA sequences and thus inhibitsinitiation in prokaryotic ribosomes. U.S. Pat. No. 5,801,153 disclosesantibiotics against pathogens. Aminoglycosides are examples ofantibacterial antibiotics that appear to inhibit protein synthesis.However, there is a limitation to their use because of their ototoxicand nephrotoxic properties. Amikacin sulfate, Framycetin sulfate,Gentamycin sulfate, Kanamycin sulfate, Neomycin sulfate, Netilmicinsulfate, Paromomycin sulfate, Sissomycin sulfate, Tobramycin, Vancomycinhydrochloride, and Viomycin sulfate are the members of theaminoglycoside family. The tools and methodologies of the presentinvention can be used to obtain derivatives of any antibiotic of choiceso that they inhibit the protein synthesis of target organisms to agreater degree than they inhibit the protein synthesis of non-targetorganisms, such as humans.

Examples of targeted and non-targeted organisms include, but are notlimited to, those provided in Table 18. TABLE 18 Examples of Classes ofMolecules which can be Identified and/or Developed by the Methods of theInvention and Applicable Target/Non-Target Organisms. Target Non-TargetType of Molecule Organisms Organisms Herbicides DicotyledonousMonocotyledonous plants plants Herbicides Grasses Soybeans, potatoes,coffee Insecticides Flies, Mites Honey bees Pesticides Ticks DeerPesticides Lice Birds Miticides Parasitic mites Dogs (mange)Antimicrobial Agents Streptococcus Humans (Antibacterials) pneumoniaeAntimicrobial Agents Clostridium Escherichia coli (Antibacterials)difficile Antimicrobial Agents Erysiphe graminis Barley (Antifungals)Antimicrobial Agents Toxoplasma gondii Animals (Antiprotozoals) PoisonsRats Dogs, cats, humans (Rodentcides)

It is contemplated that the tools and methodologies of the presentinvention can be used to obtain inhibitors of protein synthesis oftarget insects, such as bollworms and mosquitoes, more than they inhibitthe protein synthesis of non-target insects, such as beetles of thefamily Coccinellidae (e.g., ladybugs) and Apis mellifera (honey bees).Other possible target insects include, but are not limited to, insectsselected from the orders Coleoptera (beetles), Diptera (flies,mosquitoes), Hymenoptera(wasps, ants, sawflies), Lepidoptera(butterfliesand moths), Mallophaga (lice), Homoptera(whiteflies, aphids), Hemiptera(bugs), Orthroptera (locusts, cockroaches), Thysanoptera (thrips),Dermaptera(earwigs), Isoptera, Anoplura, Siphonaptera, and Trichoptera(caddis flies).

Furthermore, it is contemplated that the tools and methodologies of thepresent invention can be used to obtain inhibitors of protein synthesisof target plants which inhibit protein synthesis of the target plantsmore than they inhibit the protein synthesis of non-target plants andanimals. The target plants can be any unwanted plant species, particularweeds, and even more particularly noxious weeds. Whether or not aparticular plant is considered a weed will depend upon the context inwhich it is growing. For example, unwanted Zea mays (corn) plantsgrowing in a Glycine max (soybean) field could be considered unwantedweeds. Examples of weeds which are likely target plants include, but arenot limited to, Allium vineale (wild garlic), Bromus tectorum (downybrome), Triticum cylindricum (jointed goatgrass), Amaranthus spp.(pigsweed), Chenopodium album (lambsquarters), Avena fatua (wild oats),B. secalinus (cheat), Echinochloa crus-galli (barnyardgrass), Alopecurusmyosuroides (blackgrass), Setaria faberii (giant foxtail), Xanthiumstrumarium (common cocklebur), Ambrosia artemisiifolia (common ragweed),and Ipomoea spp. (morning glories). The non-target organisms can be anyplant, particularly any desirable plant, and even more particularly anycrop plant. The non-target organisms can also be any animals,particularly mammals, and even more particularly humans. In onepreferred embodiment, the tools and methodologies of the presentinvention can be used to produce protein synthesis inhibitors which killor injure one or more noxious weed species but fail to harm non-targetplants and animals.

Target bacteria of interest include, but are not limited to,Staphylococcus aureus, Streptococcus pyogenes, Streptococcus agalactiae,Streptococcus bovis, Streptococcus pneumoniae, Moraxella catarrhalis,Neisseria gonorrhoeae, Neisseria meningitides, Bacillus anthracis,Corynebacterium diphtheriae, Listeria monocytogenes, Erysipelothrixrhusiopathiae, Clostridium perfringens, Clostridium tetani, Clostridiumdifficile, Eschericia coli, Proteus mirabilis, Psuedomonas aeruginosa,Klebsiella pneumoniae, Haemophilus influenzae, Haemophilus ducreyi,Yersinia pestis, Yersinia enterocolitica, Francisella tularensis,Pasteurella inultocida, Vibrio cholerae, Flavobacterium meningosepticum,Pseudomonas mallei, Pseudomonas pseudomallei, Campylobacter jejuni,Campylobacter fetus, Fuso bacterium nucleatum, Calymmatobacteriumgranulomatis, Streptobacillus moniliformis, Legionella pneumophila,Mycobacterium avium-intracellulare, Mycobacterium tuberculosis,Mycobacterium leprae, Treponema pallidum, Treponema pertenue, Borreliaburgdorferi, Borrelia recurrentis, Actinomyces isrealii, Nocardiaasteroides, Ureaplasma urealyticum, Mycoplasma pneumoniae, Chlamydiapsittaci, Chlamydia trachomatis, Chlamydia pnemoniae, Pneumocystiscarinii, Coccidioides immitis, Histoplasma capsulatum, Blastomycesdermatitidis, Paracoccidioides brasiliensis, Sporothrix schenckii,Cryptococcus neoformans.

Once a candidate molecule has been designed or selected by the abovemethods, the affinity with which that molecule may bind to the ribosomeor ribosomal subunit may be tested and optimized by computationalevaluation and/or by testing biological activity after synthesizing thecompound. Candidate molecules may interact with the ribosomes orribosomal subunits in more than one conformation each of which has asimilar overall binding energy. In those cases, the deformation energyof binding may be considered to be the difference between the energy ofthe free molecule and the average energy of the conformations observedwhen the molecule binds to the ribosomes or ribosomal subunits, morepreferably to the large ribosomal subunits, and even more preferably tothe 50S ribosomal subunits.

A molecule designed or selected as binding to a ribosome or ribosomalsubunit may be further computationally optimized so that in its boundstate it preferably lacks repulsive electrostatic interaction with thetarget region. Such non-complementary (e.g., electrostatic) interactionsinclude repulsive charge-charge, dipole-dipole and charge-dipoleinteractions. Specifically, the sum of all electrostatic interactionsbetween the inhibitor and the enzyme when the inhibitor is bound to theribosome or the ribosomal subunit, preferably make a neutral orfavorable contribution to the enthalpy of binding. Weak bindingcompounds can also be designed by these methods so as to determine SAR.

Specific computer programs that can evaluate a compound deformationenergy and electrostatic interaction are available in the art. Examplesof suitable programs include: Gaussian 92, revision C (M. J. Frisch,Gaussian, Inc., Pittsburgh, Pa.); AMBER, version 4.0 (P. A. Kollman,University of California at San Francisco, Calif.); QUANTA/CHARMM(Molecular Simulations, Inc., Burlington, Mass.); and InsightII/Discover (Biosysm Technologies Inc., San Diego, Calif.). Theseprograms may be implemented, for instance, using a Silicon Graphicsworkstation, IRIS 4D/35 or IBM RISC/6000 workstation model 550. Otherhardware systems and software packages are known to those skilled in theart.

Once a molecule of interest has been selected or designed, as describedabove, substitutions may then be made in some of its atoms or sidegroups in order to improve or modify its binding properties. Generally,initial substitutions are conservative, i.e., the replacement group willapproximate the same size, shape, hydrophobicity and charge as theoriginal group. It should, of course, be understood that componentsknown in the art to alter conformation should be avoided. Suchsubstituted chemical compounds may then be analyzed for efficiency offit to the ribosome or ribosomal subunit by the same computer methodsdescribed in detail, above.

In addition, the actual ribosome-related ligands, complexes or mimeticsmay be crystallized and analyzed using X-ray diffraction. Thediffraction pattern co-ordinates are similarly used to calculate thethree-dimensional interaction of a ligand and the ribosome, ribosomalsubunit, or a mimetic, in order to confirm that the ligand binds to, orchanges the conformation of, a particular site on the ribosome orribosomal subunit, or where the mimetic has a similar three-dimensionalstructure to that of a ribosome, ribosomal subunit or a fragmentthereof.

3. Synthesis of Lead Molecules

A lead molecule of the present invention can be, but is not limited to,at least one selected from a lipid, nucleic acid, peptide, small organicor inorganic molecule, chemical compound, element, saccharide, isotope,carbohydrate, imaging agent, lipoprotein, glycoprotein, enzyme,analytical probe, and an antibody or fragment thereof, any combinationof any of the foregoing, and any chemical modification or variant of anyof the foregoing. In addition, a lead molecule may optionally comprise adetectable label. Such labels include, but are not limited to, enzymaticlabels, radioisotope or radioactive compounds or elements, fluorescentcompounds or metals, chemiluminescent compounds and bioluminescentcompounds. Well known methods may be used for attaching such adetectable label to a lead molecule.

Methods useful for synthesizing lead molecules such as lipids, nucleicacids, peptides, small organic or inorganic molecules, chemicalcompounds, elements, saccharides, isotopes, carbohydrates, imagingagents, lipoproteins, glycoproteins, enzymes, analytical probes,antibodies, and antibody fragments are well known in the art. Suchmethods include the traditional approach of synthesizing one such leadmolecule, such as a single defined peptide, at a time, as well ascombined synthesis of multiple lead molecules in a one or morecontainers. Such multiple lead molecules may include one or morevariants of a previously identified lead molecule. Methods for combinedsynthesis of multiple lead molecules are particularly useful inpreparing combinatorial libraries, which may be used in screeningtechniques known in the art.

By way of example, it is well known in the art that multiple peptidesand oligonucleotides may be simultaneously synthesized. Lead moleculesthat are small peptides up to 50 amino acids in length, may besynthesized using standard solid-phase peptide synthesis procedures, forexample, procedures similar to those described in Merrifield (1963) J.Am. Chem. Soc., 85: 2149. For example, during synthesis, N-α-protectedamino acids having protected side chains are added stepwise to a growingpolypeptide chain linked by its C-terminal end to an insoluble polymericsupport, e.g., polystyrene beads. The peptides are synthesized bylinking an amino group of an N-α-deprotected amino acid to an α-carboxygroup of an N-α-protected amino acid that has been activated by reactingit with a reagent such as dicyclohexylcarbodiimide. The attachment of afree amino group to the activated carboxyl leads to peptide bondformation. The most commonly used N-α-protecting groups include Bocwhich is acid labile and Fmoc which is base labile.

Briefly, the C-terminal N-α-protected amino acid is first attached tothe polystyrene beads. Then, the N-α-protecting group is removed. Thedeprotected α-amino group is coupled to the activated α-carboxylategroup of the next N-α-protected amino acid. The process is repeateduntil the desired peptide is synthesized. The resulting peptides arecleaved from the insoluble polymer support and the amino acid sidechains deprotected. Longer peptides, for example greater than about 50amino acids in length, typically are derived by condensation ofprotected peptide fragments. Details of appropriate chemistries, resins,protecting groups, protected amino acids and reagents are well known inthe art and so are not discussed in detail herein. See for example,Atherton et al. (1963) Solid Phase Peptide Synthesis: A PracticalApproach (IRL Press), and Bodanszky (1993) Peptide Chemistry, APractical Textbook, 2nd Ed. Springer-Verlag, and Fields et al. (1990)Int. J. Peptide Protein Res. 35:161-214.

Purification of the resulting peptide is accomplished using conventionalprocedures, such as preparative HPLC, e.g., gel permeation, partitionand/or ion exchange chromatography. The choice of appropriate matricesand buffers are well known in the art and so are not described in detailherein.

It is contemplated that a synthetic peptide in accordance with theinvention may comprise naturally occurring amino acids, unnatural aminoacids, and/or amino acids having specific characteristics, such as, forexample, amino acids that are positively charged, negatively charged,hydrophobic, hydrophilic, or aromatic. As used herein, the term“naturally occurring amino acids” refers to the L-isomers of amino acidsnormally found in proteins. The predominant naturally occurring aminoacids are glycine, alanine, valine, leucine, isoleucine, serine,methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine,proline, histidine, aspartic acid, asparagine, glutamic acid,glutarmine, arginine, and lysine. Unless specifically indicated, allamino acids are referred to in this application are in the L-form.Furthermore, as used herein, the term “unnatural amino acids” refers toamino acids that are not naturally found in proteins. For example,selenomethionine.

Amino acids that are “positively charged” include any amino acid havinga positively charged side chain under normal physiological conditions.Examples of positively charged naturally occurring amino acids include,for example, arginine, lysine, and histidine. Conversely, amino acidsthat are “negatively charged” include any amino acid having a negativelycharged side chains under normal physiological conditions. Examples ofnegatively charged naturally occurring amino acids include, for example,aspartic acid and glutamic acid.

As used herein, the term “hydrophobic amino acid” includes any aminoacids having an uncharged, nonpolar side chain that is relativelyinsoluble in water. Examples of naturally occurring hydrophobic aminoacids include, for example, alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine. In addition, as usedherein, the term “hydrophilic amino acid” refers to any amino acidshaving an uncharged, polar side chain that is relatively soluble inwater. Examples of naturally occurring hydrophilic amino acids include,for example, serine, threonine, tyrosine, asparagine, glutamine andcysteine.

Finally, as used herein, the term “aromatic” refers to amino acidresidues which side chains have delocalized conjugated system. Examplesof aromatic residues include, for example, phenylalanine, tryptophan,and tyrosine.

With regard to the production of non-peptide small organic moleculeswhich act as a ligand in the present invention, these molecules can besynthesized using standard organic chemistries well known and thoroughlydocumented in the patent and other literatures.

Many of the known methods useful in synthesizing lead of the presentinvention may be automated, or may otherwise be practiced on acommercial scale. As such, once a lead molecule has been identified ashaving commercial potential, mass quantities of that molecule may easilybe produced.

4. Characterization of Molecules

Molecules designed, selected and/or optimized by methods describedabove, once produced, may be characterized using a variety of assaysknown to those skilled in the art to determine whether the compoundshave biological activity. For example, the molecules may becharacterized by conventional assays, including but not limited to thoseassays described below, to determine whether they have a predictedactivity, binding activity and/or binding specificity.

Furthermore, high-throughput screening may be used to speed up analysisusing such assays. As a result, it may be possible to rapidly screen newmolecules for their ability to interact with a ribosome or ribosomalsubunit using the tools and methods of the present invention. Generalmethodologies for performing high-throughput screening are described,for example, in Devlin, (1998), High Throughput Screening, MarcelDekker; and U.S. Pat. No. 5,763,263. High-throughput assays can use oneor more different assay techniques including, but not limited to, thosedescribed below.

(1) Surface Binding Studies. A variety of binding assays may be usefulin screening new molecules for their binding activity. One approachincludes surface plasmon resonance (SPR) which can be used to evaluatethe binding properties molecules of interest with respect to a ribosome,ribosomal subunit or a fragment thereof.

SPR methodologies measure the interaction between two or moremacromolecules in real-time through the generation of aquantum-mechanical surface plasmon. One device, (BlAcore Biosensor RTMfrom Pharmacia Biosensor, Piscatawy, N.J.) provides a focused beam ofpolychromatic light to the interface between a gold film (provided as adisposable biosensor “chip”) and a buffer compartment that can beregulated by the user. A 100 nm thick “hydrogel” composed ofcarboxylated dextran which provides a matrix for the covalentimmobilization of analytes of interest is attached to the gold film.When the focused light interacts with the free electron cloud of thegold film, plasmon resonance is enhanced. The resulting reflected lightis spectrally depleted in wavelengths that optimally evolved theresonance. By separating the reflected polychromatic light into itscomponent wavelengths (by means of a prism), and determining thefrequencies which are depleted, the BIAcore establishes an opticalinterface which accurately reports the behavior of the generated surfaceplasmon resonance. When designed as above, the plasmon resonance (andthus the depletion spectrum) is sensitive to mass in the evanescentfield (which corresponds roughly to the thickness of the hydrogel). Ifone component of an interacting pair is immobilized to the hydrogel, andthe interacting partner is provided through the buffer compartment, theinteraction between the two components can be measured in real timebased on the accumulation of mass in the evanescent field and itscorresponding effects of the plasmon resonance as measured by thedepletion spectrum. This system permits rapid and sensitive real-timemeasurement of the molecular interactions without the need to labeleither component.

(2) Immunodiagnostics and Immunoassays. These are a group of techniquesthat can be used for the measurement of specific biochemical substances,commonly at low concentrations in complex mixtures such as biologicalfluids, that depend upon the specificity and high affinity shown bysuitably prepared and selected antibodies for their complementaryantigens. A substance to be measured must, of necessity, beantigenic—either an immunogenic macromolecule or a haptenic smallmolecule. To each sample a known, limited amount of specific antibody isadded and the fraction of the antigen combining with it, often expressedas the bound:free ratio, is estimated, using as indicator a form of theantigen labeled with radioisotope (radioimmunoassay), fluorescentmolecule (fluoroimmunoassay), stable free radical (spin immunoassay),enzyme (enzyme immunoassay), or other readily distinguishable label.

Antibodies can be labeled in various ways, including: enzyme-linkedimmunosorbent assay (ELISA); radioimmuno assay (RIA); fluorescentimmunoassay (FIA); chemiluminescent immunoassay (CLIA); and labeling theantibody with colloidal gold particles (immunogold).

Common assay formats include the sandwich assay, competitive orcompetition assay, latex agglutination assay, homogeneous assay,microtitre plate format and the microparticle-based assay.

(3) Enzyme-linked immunosorbent assay (ELISA). ELISA is animmunochemical technique that avoids the hazards of radiochemicals andthe expense of fluorescence detection systems. Instead, the assay usesenzymes as indicators. ELISA is a form of quantitative immunoassay basedon the use of antibodies (or antigens) that are linked to an insolublecarrier surface, which is then used to “capture” the relevant antigen(or antibody) in the test solution. The antigen-antibody complex is thendetected by measuring the activity of an appropriate enzyme that hadpreviously been covalently attached to the antigen (or antibody).General methods and compositions for practicing ELISA are described, forexample, in Crowther (1995) ELISA—Theory and Practice (Methods inMolecular Biology), Humana Press; Challacombe and Kemeny, (1998) ELISAand Other Solid Phase Immunoassays—Theoretical and Practical Aspects,John Wiley; Kemeny, (1991) A Practical Guide to ELISA, Pergamon Press;Ishikawa, (1991) Ultrasensitive and Rapid Enzyme Immunoassay (LaboratoryTechniques in Biochemistry and Molecular Biology) Elsevier.

(4) Colorimetric Assays. Colorimetry is any method of quantitativechemical analysis in which the concentration or amount of a compound isdetermined by comparing the color produced by the reaction of a reagentwith both standard and test amounts of the compound, often using acolorimeter. A calorimeter is a device for measuring color intensity ordifferences in color intensity, either visually or photoelectrically.

Standard colorimetric assays of beta-galactosidase enzymatic activityare well known to those skilled in the art (see, for example, Norton etal. (1985) Mol. Cell. Biol. 5: 281-290). A calorimetric assay can beperformed on whole cell lysates usingO-nitrophenyl-β-D-galactopyranoside (ONPG, Sigma) as the substrate in astandard colorimetric beta-galactosidase assay (Sambrook et al. (1989)Molecular Cloning—A Laboratory Manual, Cold Spring Harbor LaboratoryPress). Automated colorimetric assays are also available for thedetection of β-galactosidase activity, as described in U.S. Pat. No.5,733,720.

(5) Immunofluorescence Assays. Immunofluorescence or immunofluorescencemicroscopy is a technique in which an antigen or antibody is madefluorescent by conjugation to a fluorescent dye and then allowed toreact with the complementary antibody or antigen in a tissue section orsmear. The location of the antigen or antibody can then be determined byobserving the fluorescence by microscopy under ultraviolet light.

A general description of immunofluorescent techniques appears forexample, in Knapp et al. (1978) Immunofluorescence and Related StainingTechniques, Elsevier; Allan, (1999) Protein Localization by FluorescentMicroscopy—A Practical Approach (The Practical Approach Series) OxfordUniversity Press; Caul, (1993) Immunofluorescence Antigen DetectionTechniques in Diagnostic Microbiology, Cambridge University Press. Fordetailed explanations of immunofluorescent techniques applicable to thepresent invention, see, for example, U.S. Pat. No. 5,912,176; U.S. Pat.No. 5,869,264; U.S. Pat. No. 5,866,319; and U.S. Pat. No. 5,861,259.

(6) Fluorescence Polarization. Fluorescence polarization (FP) is ameasurement technique that can readily be applied to protein-protein andprotein-ligand interactions in order to derive IC₅₀s and Kds of theassociation reaction between two molecules. In this technique one of themolecules of interest is conjugated with a fluorophore. This isgenerally the smaller molecule in the system (in this case, the moleculeof interest). The sample mixture, containing both the ligand-probeconjugate and the ribosome, ribosomal subunit or fragment thereof, isexcited with vertically polarized light. Light is absorbed by the probefluorophores, and re-emitted a short time later. The degree ofpolarization of the emitted light is measured. Polarization of theemitted light is dependent on several factors, but most importantly onviscosity of the solution and on the apparent molecular weight of thefluorophore. With proper controls, changes in the degree of polarizationof the emitted light depends only on changes in the apparent molecularweight of the fluorophore, which in-turn depends on whether theprobe-ligand conjugate is free in solution, or is bound to a receptor.Binding assays based on FP have a number of important advantages,including the measurement of IC₅₀s and Kds under true homogenousequilibrium conditions, speed of analysis and amenity to automation, andability to screen in cloudy suspensions and colored solutions.

(7) Protein Synthesis. It is contemplated that, in addition tocharacterization by the foregoing biochemical assays, the molecule ofinterest may also be characterized as a modulator (for example, aninducer of protein synthesis or an inhibitor of protein synthesis) ofthe functional activity of the ribosome or ribosomal subunit.

Inhibitors of protein synthesis may be assayed on the cellular level.For example, molecules of interest can be assayed for inhibitory actionagainst organisms, for example, micro-organism, by growing themicro-organism of interest in media either containing or lacking themolecule of interest. Growth inhibition may be indicative that themolecule may be acting as a protein synthesis inhibitor.

Furthermore, more specific protein synthesis inhibition assays may beperformed by administering the compound to a whole organism, tissue,organ, organelle, cell, a cellular or subcellular extract, or a purifiedribosome preparation and observing its pharmacological and inhibitoryproperties by determining, for example, its inhibition constant (IC₅₀)for inhibiting protein synthesis. Incorporation of ³H leucine or ³⁵Smethionine, or similar experiments can be performed to investigateprotein synthesis activity.

A change in the amount or the rate of protein synthesis in the cell inthe presence of a molecule of interest indicates that the molecule is aninducer of protein synthesis. A decrease in the rate or the amount ofprotein synthesis indicates that the molecule is a inhibitor of proteinsynthesis.

H. Drug Formulation and Administration

It is contemplated that once identified, the active molecules of theinvention may be incorporated into any suitable carrier prior to use.More specifically, the dose of active molecule, mode of administrationand use of suitable carrier will depend upon the target and non-targetorganism of interest.

It is contemplated that with regard to mammalian recipients, thecompounds of interest may be administered by any conventional approachknown and/or used in the art. Thus, as appropriate, administration canbe oral or parenteral, including intravenous and intraperitoneal routesof administration. In addition, administration can be by periodicinjections of a bolus, or can be made more continuous by intravenous orintraperitoneal administration from a reservoir which is external (e.g.,an intrvenous bag). In certain embodiments, the compounds of theinvention can be therapeutic-grade. That is, certain embodiments complywith standards of purity and quality control required for administrationto humans. Veterinary applications are also within the intended meaningas used herein.

The formulations, both for veterinary and for human medical use, of thedrugs according to the present invention typically include such drugs inassociation with a pharmaceutically acceptable carrier therefore andoptionally other therapeutic ingredient(s). The carrier(s) should be“acceptable” in the sense of being compatible with the other ingredientsof the formulations and not deleterious to the recipient thereof.Pharmaceutically acceptable carriers, in this regard, are intended toinclude any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is known in theart. Except insofar as any conventional media or agent is incompatiblewith the active compound, use thereof in the compositions iscontemplated. Supplementary active compounds (identified or designedaccording to the invention and/or known in the art) also can beincorporated into the compositions. The formulations may conveniently bepresented in dosage unit form and may be prepared by any of the methodswell known in the art of pharmacy/microbiology. In general, someformulations are prepared by bringing the drug into association with aliquid carrier or a finely divided solid carrier or both, and then, ifnecessary, shaping the product into the desired formulation.

A pharmaceutical composition of the invention should be formulated to becompatible with its intended route of administration. Examples of routesof administration include oral or parenteral, e.g., intravenous,intradermal, inhalation, transdermal (topical), transmucosal, and rectaladministration. Solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide.

Useful solutions for oral or parenteral administration can be preparedby any of the methods well known in the pharmaceutical art, described,for example, in Remington's Pharmaceutical Sciences, (Gennaro, A., ed.),Mack Pub., (1990). Formulations for parenteral administration can alsoinclude glycocholate for buccal administration, methoxysalicylate forrectal administration, or cutric acid for vaginal administration. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic. Suppositories forrectal administration also can be prepared by mixing the drug with anon-irritating excipient such as cocoa butter, other glycerides, orother compositions which are solid at room temperature and liquid atbody temperatures. Formulations also can include, for example,polyalkylene glycols such as polyethylene glycol, oils of vegetableorigin, hydrogenated naphthalenes, and the like. Formulations for directadministration can include glycerol and other compositions of highviscosity. Other potentially useful parenteral carriers for these drugsinclude ethylene-vinyl acetate copolymer particles, osmotic pumps,implantable infusion systems, and liposomes. Formulations for inhalationadministration can contain as excipients, for example, lactose, or canbe aqueous solutions containing, for example, polyoxyethylene-9-laurylether, glycocholate and deoxycholate, or oily solutions foradministration in the form of nasal drops, or as a gel to be appliedintranasally. Retention enemas also can be used for rectal delivery.

Formulations of the present invention suitable for oral administrationmay be in the form of discrete units such as capsules, gelatin capsules,sachets, tablets, troches, or lozenges, each containing a predeterminedamount of the drug; in the form of a powder or granules; in the form ofa solution or a suspension in an aqueous liquid or non-aqueous liquid;or in the form of an oil-in-water emulsion or a water-in-oil emulsion.The drug may also be administered in the form of a bolus, electuary orpaste. A tablet may be made by compressing or moulding the drugoptionally with one or more accessory ingredients. Compressed tabletsmay be prepared by compressing, in a suitable machine, the drug in afree-flowing form such as a powder or granules, optionally mixed by abinder, lubricant, inert diluent, surface active or dispersing agent.Moulded tablets may be made by moulding, in a suitable machine, amixture of the powdered drug and suitable carrier moistened with aninert liquid diluent.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients. Oral compositions preparedusing a fluid carrier for use as a mouthwash include the compound in thefluid carrier and are applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose; a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition should be sterile and should be fluid to theextent that easy syringability exists. It should be stable under theconditions of manufacture and storage and should be preserved againstthe contaminating action of microorganisms such as bacteria and fungi.The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyetheylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation include vacuumdrying and freeze-drying which yields a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Formulations suitable for intra-articular administration may be in theform of a sterile aqueous preparation of the drug which may be inmicrocrystalline form, for example, in the form of an aqueousmicrocrystalline suspension. Liposomal formulations or biodegradablepolymer systems may also be used to present the drug for bothintra-articular and ophthalmic administration.

Formulations suitable for topical administration, including eyetreatment, include liquid or semi-liquid preparations such as liniments,lotions, gels, applicants, oil-in-water or water-in-oil emulsions suchas creams, ointments or pasts; or solutions or suspensions such asdrops. Formulations for topical administration to the skin surface canbe prepared by dispersing the drug with a dermatologically acceptablecarrier such as a lotion, cream, ointment or soap. Particularly usefulare carriers capable of forming a film or layer over the skin tolocalize application and inhibit removal. For topical administration tointernal tissue surfaces, the agent can be dispersed in a liquid tissueadhesive or other substance known to enhance adsorption to a tissuesurface. For example, hydroxypropylcellulose or fibrinogen/thrombinsolutions can be used to advantage. Alternatively, tissue-coatingsolutions, such as pectin-containing formulations can be used.

For inhalation treatments, inhalation of powder (self-propelling orspray formulations) dispensed with a spray can, a nebulizer, or anatomizer can be used. Such formulations can be in the form of a finepowder for pulmonary administration from a powder inhalation device orself-propelling powder-dispensing formulations. In the case ofself-propelling solution and spray formulations, the effect may beachieved either by choice of a valve having the desired spraycharacteristics (i.e., being capable of producing a spray having thedesired particle size) or by incorporating the active ingredient as asuspended powder in controlled particle size. For administration byinhalation, the compounds also can be delivered in the form of anaerosol spray from pressured container or dispenser which contains asuitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration also can be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants generally are known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfilsidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds typically areformulated into ointments, salves, gels, or creams as generally known inthe art.

The active compounds may be prepared with carriers that will protect thecompound against rapid elimination from the body, such as a controlledrelease formulation, including implants and microencapsulated deliverysystems. Biodegradable, biocompatible polymers can be used, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations will be apparent to those skilled in the art. The materialsalso can be obtained commercially from Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811. Microsomes and microparticles also can be used.

Oral or parenteral compositions can be formulated in dosage unit formfor ease of administration and uniformity of dosage. Dosage unit formrefers to physically discrete units suited as unitary dosages for thesubject to be treated; each unit containing a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

As noted above, drugs identified or designed according to the inventioncan be formulated into pharmaceutical compositions by admixture withpharmaceutically acceptable nontoxic excipients and carriers. Suchcompositions can be prepared for parenteral administration, particularlyin the form of liquid solutions or suspensions; for oral administration,particularly in the form of tablets or capsules; or intranasally,particularly in the form of powders, nasal drops or aerosols. Whereadhesion to a tissue surface is desired the composition can include thedrug dispersed in a fibrinogen thrombin composition or otherbioadhesive. The drug then can be painted, sprayed or otherwise appliedto the desired tissue surface. Alternatively, the drugs can beformulated for parenteral or oral administration to humans or othermammals, for example, in therapeutically effective amounts, e.g.,amounts which provide appropriate concentrations of the drug to targettissue for a time sufficient to induce the desired effect.

Where the active compound is to be used as part of a transplantprocedure, it can be provided to the living tissue or organ to betransplanted prior to removal of tissue or organ from the donor. Thedrug can be provided to the donor host. Alternatively or, in addition,once removed from the donor, the organ or living tissue can be placed ina preservation solution containing the active compound. In all cases,the active compound can be administered directly to the desired tissue,as by injection to the tissue, or it can be provided systemically,either by oral or parenteral administration, using any of the methodsand formulations described herein and/or known in the art.

Where the drug comprises part of a tissue or organ preservationsolution, any commercially available preservation solution can be usedto advantage. For example, useful solutions known in the art includeCollins solution, Wisconsin solution, Belzer solution, Eurocollinssolution and lactated Ringer's solution.

The effective concentration of the compounds to be delivered in atherapeutic composition will vary depending upon a number of factors,including the final desired dosage of the compound to be administeredand the route of administration. The preferred dosage to be administeredalso is likely to depend on such variables as the type and extent ofdisease or indication to be treated, the overall health status of theparticular patient, the relative biological efficacy of the compounddelivered, the formulation of the drug, the presence and types ofexcipients in the formulation, and the route of administration. Ingeneral terms, the drugs of this invention can be provided to anindividual using typical dose units deduced from the earlier-describedmammalian studies using non-human primates and rodents.

When the active compounds are nucleic acid molecules, the nucleic acidmay be inserted into vectors and used as gene therapy vectors. Genetherapy vectors can be delivered to a subject by, for example,intravenous injection, local administration (see U.S. Pat. No.5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994)Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparationof the gene therapy vector can include the gene therapy vector in anacceptable diluent, or can comprise a slow release matrix in which thegene delivery vehicle is imbedded. Alternatively, where the completegene delivery vector can be produced intact from recombinant cells, e.g.retroviral vectors, the pharmaceutical preparation can include one ormore cells which produce the gen delivery system.

When an active compound of the invention is intended for administrationto a plant host, the invention may be applied directly to the plantenvironment, for example, to the surface of leaves, buds, roots orfloral parts. Alternatively, the present invention can be used as a seedcoating. The determination of an effective amount of the presentinvention as required for a particular plant is within the skill of theart and will depend on such factors as the plant species, method ofplanting, and soil type. It is contemplated that compositions containingdrugs of the invention can be prepared by formulating such drugs withadjuvants, diluents, carriers, etc., to provide compositions in the formof filings/divided particulate solids, granules, pellets, wetablepowders, dust, aqueous suspensions or dispersions, and emulsions. It isfurther contemplated to use such drugs in capsulated form, for example,the drugs can be encapsulated within polymer, gelatin, lipids or otherformulation aids such as emulsifiers, surfactants wetting agents,antifoam agents and anti-freeze agents, may be incorporated into suchcompositions especially if such compositions will be stored for anyperiod of time prior to use. Application of compositions containingdrugs of the invention as the active agent can be carried out byconventional techniques. When an active compound is intended foradministration to an insect host, standard methods such as, but notlimited to, aerial dispersal are contemplated.

Active compound identified or designed by a method of the invention alsoinclude precursors of the active compounds. The term precursors refersto a pharmacologically inactive (or partially inactive) derivative of aparent molecule that requires biotransformation, either spontaneous orenzymatic, within the organism to release the active compounds.Precursors are variations or derivatives of the compounds of theinvention which have groups cleavable under metabolic conditions.Precursors become the active compounds of the invention which arepharmaceutically active in vivo, when they undergo solvolysis underphysiological conditions or undergo enzymatic degradation. Precursorforms often offer advantages of solubility, tissue compatibility, ordelayed release in the mammalian organism (see Bundgard, Design ofProdrugs, pp. 7-9, 21-24, Elsevier, Amsterdam (1985); and Silverman, TheOrganic Chemistry of Drug Design and Drug Action, pp. 352-401, AcademicPress, San Diego, Calif. (1992).

Active compound as identified or designed by the methods describedherein can be administered to individuals to treat disorders(prophylactically or therapeutically). In conjunction with suchtreatment, pharmacogenomics (i.e., the study of the relationship betweenan individual's genotype and that individual's response to a foreigncompound or drug) may be considered. Differences in metabolism oftherapeutics can lead to severe toxicity or therapeutic failure byaltering the relation between dose and blood concentration of thepharmacologically active drug. Thus, a physician or clinician mayconsider applying knowledge obtained in relevant pharmacogenomicsstudies in determining whether to administer a drug as well as tailoringthe dosage and/or therapeutic regimen of treatment with the drug.

With regard to mammals, it is contemplated that the effective dose of aprotein synthesis inducer or inhibitor will be in the range of about0.01 to about 50 mg/kg, preferably about 0.1 to about 10 mg/kg of bodyweight, administered in single or multiple doses. Typically, the induceror inhibitor may be administered to a human recipient in need oftreatment at a daily dose range of about 1 to about 2000 mg per patient.

In light of the foregoing general discussion, the specific examplespresented below are illustrative only and are not intended to limit thescope of the invention. Other generic and specific configurations willbe apparent to those persons skilled in the art.

III. EXAMPLES A. Example 1 Preparation of 50S Ribosomal Subunit Crystals

H. marismortui (ATCC 43049) was grown as described previously (Ban etal. (1998) supra) on a slightly modified version of ATCC culture medium1230, which was supplemented with 4.3 g of yeast extract, 5.1 g of Tris,and 3.4 g of glucose per liter. Bacteria were grown at 37° C. to anOD₅₅₀ nm between 1.0 and 2.2. They were harvested by centrifugation, andstored at −80° C. Cells were ruptured using a French press. Ribosomeswere prepared from lysates by centrifugation, and subunits were isolatedon sucrose gradients (Shevack et al. (1985) FEBS Lett. 184: 68-71).

1. Reverse Extraction

(1) Take 1 mg of subunits from a concentrated 50S ribosomal subunitstock (30 mg/ml in 1.2 M KCl, 0.5 M NH4Cl, 20 mM MgCl₂, Tris 10 mM,CdCl₂ 1 mM, Tris 5 mM, pH 7.5) and mix with ½ vol. of 30% PEG6000 (300gPEG, 700 ml H₂O to make 1 liter of 30% PEG; filter through 0.2 μmfilter). Leave on ice for 1 to 2 hr.

(2) Spin down precipitate for about 30 seconds using a desktopcentrifuge.

(3) Remove supernatant and add 100 μl of RE-buffer: 7% PEG6000, 1.2 MKCl, 0.5 M NH₄Cl, 100 mM KAc, 30 mM MgCl₂, 10 mM Tris, 10 mM MES (pH7.5), and 1 mM CdCl₂.

(4) Resuspend pellet at room temperature by mixing with a P200 pipetteset at 50 μl. Resuspended material should appear a little cloudy.

(5) Wrap the eppendorf tube in aluminum foil and leave for equilibrationat room temperature for 30-60 min. The solution will be saturated with50S.

(6) Spin down for 2 minutes in desk-top centrifuge at room temperature,transfer supernatant to new eppendorf tube. A little pellet should befound in the tube used for centrifugation. Keep the supernatant at roomtemperature.

(7) Put 8-10 μl of supernatant in the sample well of a sitting drop tray(Charles-Supper). Streak seed one hour later from a seed stock. Seedstock is prepared by putting previously grown crystals in stabilizingsolution buffer A (see below), and then vortexing them violently. Tostreak seed, a human hair cleaned with water and ethanol and then driedis passed through the vortexed solution and then touched on the newcrystallization drop. Drops should look cloudy. The reservoirs in thesitting drop trays contain 1000 μl of a solution containing 8% PEG6000,1.2 M KCl, 0.5 M NH₄Cl, 100 mM KAc, 6.5 mM HAc (yields pH 5.8), 30 mMMgCl₂, and 1 mM CdCl₂.

(8) Check after one day if seeding is succeeded. If yes, let crystalsgrow for three weeks.

2. Stabilization Protocol

When crystals have finished growing (after approximately 3 weeks), eachsitting drop chamber is opened by making just a single cut (slit) goingfrom the middle and to the edge of the well. Through this narrow slit,10 μl of buffer A (1.2 M KCl, 0.5 M NH₄Cl, 30 mM MgCl₂, 10% PEG6000, 1mM CdCl₂, 100 mM KAc, 10 mM Tris (titrated to final pH 6.1), 30 mM MES)at room temperature is added to each drop and 45 μl of Buffer C (0.667 MMES, 0.333 M Tris) to each reservoir.

Trays are put in a plastic box with a lid, and put in a 16° C. incubatorfor approximately one day, and then moved to 12° C. for another day. Theplastic box is then put in a polystyrene container with a lid, and putin the cold room for yet another day. Crystals can be kept like this fora long time, but need to undergo a further changing of buffer prior toany use.

Make the following transition series using buffer A and buffer B (1.7 MNaCl, 0.5 M NH₄Cl, 30 mM MgCl₂, 1 mM CdCl₂, 12% PEG6000, 20% EG, 100MMKAC (titrated to final pH 5.8 with HAC) to give final ratios of buffer Bto buffer A of: 1/16, ⅛, ¼, ½, ¾. All solutions should be at cold roomtemperature. All manipulations of the drops will take place through thenarrow slit.

(1) Add 40 μl “ 1/16” to the drop, leave for 15 minutes.

(2) Add 40 μl “⅛” to the drop, leave for 30-60 minutes.

(3) Take out 40 μl from the drop (and discard it in the reservoir), add40 μl “¼”, leave for 30-60 minutes.

(4) Take out 40 μl from the drop (and discard it in the reservoir), add40 μl “½”, leave for 15 minutes.

(5) Take out 40 μl from the drop (and discard it in the reservoir), add40 μl “¾”, leave for 15 minutes.

(6) Take out 40 μl from the drop (and discard it in the reservoir), add40 μl buffer B, leave for 15 minutes.

(7) Take out 60-80 μl from the drop (and discard it in the reservoir),add 60-80 μl Buffer B, replace reservoirs with 500 μl buffer B.

B. Example 2 Determination of the Crystal Structure of the 50S RibosomalSubunit, With the Initial Refinement

All data, except the two native data sets, were collected at theNational Synchrotron Light Source (Brookhaven) from crystals frozen at100 K, using beamlines X12b and X25 and recorded using a 345 mm MARimaging plate. For each heavy atom derivative, anomalous diffractiondata were collected at the wavelength corresponding to the peakanomalous scattering. The beam size was 100×100 μm for most datacollections at X25 and 200×200 μm at beamline X12b. The crystals werealigned along the long axis of the unit cell (570 Å) so that 1.0°oscillations could be used to collect reflections out to a maximum of2.7 Å resolution at the edge of the MAR detector. At beamline X12b thecrystal to detector distances varied between 450.0 mm and 550.0 mmdepending on wavelength, crystal quality and beam divergence, and it waschosen so that maximum resolution data could be collected while avoidingoverlapping of spots. At beamline X25 the detector was positioned on arigid platform at 480 mm which allowed data collection to 3.2 Å foriridium and osmium derivatives with the wavelength set at the anomalousedge. Native data to 2.4 Å resolution were collected at the structuralbiology beamline ID19 of the Advanced Photon Source (Argonne) using aCCD detector (etc.). Data sets were processed by using DENZO andSCALEPACK (Otwinowski, (1993) Data Collection and Processing).

Heavy atom based phasing was extended to 3.2 Å resolution by combiningMIR phases calculated for two different isomorphous groups of data (MIR1and MIR2, Table 1) with single derivative anomalous dispersion (SAD)phases. The best two derivatives were osmium pentamine and iridiumhexamine, each of which contained a large number of binding sites (Table1). Several other derivatives with smaller number of sites furtherimproved map quality. All phasing was done by maximum likelihood methodimplemented in CNS (Brunger et al. (1998) supra) with the exception ofthe Ta₆Br₁₂ derivative, which was refined in SHARP (de La Fortelle,(1997) Meth. Enzymol. 276: 472-494) represented as spherically averagedelectron density (Table 1). Phases were improved and extended from 3.3 Åto 2.4 Å by solvent flipping (Abrahams et al. (1996) supra) and modelswere built.

C. Example 3 Preparation of Crystals of 50S Ribosomal Subunit/PuromycinComplex and Collection of X-ray Diffraction Data

Crystals of 50S ribosomal subunits were grown and stabilized asdescribed earlier. CCdA-p-puromycin (see FIG. 9A) was a generous giftfrom Michael Yarus (Welch, et al. (1995) supra). Oligonucleotides fromamino-N-acylated minihelices (see FIG. 9B) were synthesized byDharmacon. Following deprotection, the oligonucleotides were heatedbriefly to 100° C. and snap-cooled on ice to reanneal. Ribosomal 50Ssubunit crystals were stabilized and then soaked for 24 hours instabilization buffer plus 100 μM CCdA-p-puromycin or amino-N-acylatedmini-helices prior to cryovitrification in liquid propane and X-raydiffraction data collection. Phases were calculated by densitymodification (CNS) beginning with the best experimental phases using2F_(o)(analogue)-F_(o)(native) for amplitudes, from 60.0 to 3.2 Å.(Native amplitudes were from the most isomorphous native 1 data set,except for those amplitudes which were present only in the more completenative 2 data set. Calculated 2F_(o)-F_(o) amplitudes which were lessthan twice the corresponding calculated & were replaced byF_(o)(analogue)). Maps were then calculated using phases from densitymodified and 2F_(o)(analogue)-F_(o)(native) orF_(o)(analogue)-F_(o)(native) amplitudes.

D. Example 4 Antibiotic Binding Sites Located in the Polypeptide ExitTunnel Near the Peptidyl Transferase Center

Crystalline complexes of the H. marismortui large subunit complexed withthree antibiotics have been established at about 3.0 Å resolution. Theelectron density maps at this resolution have allowed us to positionapproximately on the ribosome the antibiotics tylosin, carbomycin andanisomycin. We observed that these antibiotics all bind to the ribosomein the region that lies between the peptidyl transferase center asdefined by the Yarus inhibitor, CCdA-p-puromycin, and the tips of theproteins L22 and L4 at the point that they form a small orifice in thepolypeptide exit tunnel. The general location of this major antibioticbinding site is shown in FIG. 19. Tylosin and carbomycin appear tofunction by blocking the exit of newly synthesized polypeptides.Anisomycin blocks the A site.

It is contemplated that the antibiotic erythromycin will bind in almostthe same location as tylosin because of the similarity of the twomolecules and because erythromycin resistance mutations are known inboth the tip of protein L4 and in portions of the RNA near the tylosinbinding site.

The vast majority of the interactions between those antibiotics and theribosome are through rRNA that forms the A site, and the surface of thetunnel between the peptidyl transferase center and protein L22. Sincethese antibiotics do not bind identically, there will be many additionalways that small molecule compounds can be designed to bind in thisregion using the tools and methodologies of the present invention. Forexample, by connecting together components of each of the differentantibiotics which bind to non-overlapping sites it will be possible tocreate new antibiotics (see, Example 6). In addition, based on newprinciples of small molecule RNA interaction shown by these antibioticcomplexes we will be able to design entirely novel small molecules thatwill bind to sites on the ribosome as well as other potential RNAtargets.

E. Example 5 Design and Testing of Hybrid Antibiotics

Many antibiotics that target ribosomes, more particularly largeribosomal subunits, and disrupt protein synthesis are complex moleculesthat are effectively concatenations of simpler substructures, at leastone of which interacts with a discrete part of the ribosome. When thecompound in question includes several interactive substructures, itsbinding site is effectively the sum of the subsites that contact andengage each such substructure. It has been found that many antibioticsthat target the large ribosomal subunit bind the ribosomal subunit atsites that are close to one another. Thus the possibility exists ofsynthesizing new antibiotics in which one ribosome-binding moiety of afirst known antibiotic is linked chemically to a ribosome-binding moietyof a second known antibiotic that interacts with an adjacent subsite.The new compound that results is thus a chimera of the two antibioticsfrom which it derives.

Chimeric antibiotics can be designed using the information about thestructures of antibiotic/ribosome complexes discussed hereinabove. Thesestructures permit the identification of antibiotic binding subsites inthe ribosome, and the specification of the chemical entities thatinteract with them. Equipped with such knowledge, those skilled in theart of organic synthesis can synthesize compounds that link thesubstructures of interest together in ways that should enable them tointeract with their respective subsites at the same time. Any compounddevised this way that functions in the manner intended is likely toinhibit cell growth and if it does, protein synthesis in vivo. At thevery least, it should block protein synthesis in in-vitro assay systems.Further information about the ribosomal interactions of such a compoundcan be obtained by determining the structure of the complex it formswith the ribosome using the methods described in Section D, hereinabove.

For example, as a result of the work described herein, it has beendiscovered that the disaccharide moiety of carbomycin binds the largeribosomal subunit at a site in close proximity to the binding site for aportion of the anisomycin. Using this information and the softwarepackages described hereinabove, the skilled artisan can design a hybridantibiotic comprising the relevant ribosome binding portions ofcarbomycin and anisomycin linked by a suitable chemical linker. Thishybrid molecule, once designed, can be synthesized and purified usingconventional synthetic organic chemistries and conventional purificationschemes. Once synthesized and purified, the hybrid molecule can bescreened for bioactivity. These screens can include, for example,growing micro-organisms on or in media either supplemented or lackingthe hybrid molecule. Any reduction in the number of micro-organisms orthe size of colonies in the presence of the hybrid molecule would beindicative of bioactivity. Furthermore, the hybrid molecule could betested in a cell free translation system in the presence of one or morelabeled amino acids. Any reduction in the level of labeled amino acidsincorporated into proteins in cell free systems that include the hybridmolecule relative to cell free systems locking the hybrid molecule wouldbe indicative that the hybrid molecule acts as a functional proteinsynthesis inhibitor. It is contemplated that the hybrid molecule couldthen be iteratively refined as discussed hereinabove to enhance itsbioactive peptides and bioavailability.

Incorporation by Reference

The disclosure of each of the patent documents, scientific articles,atomic-co-ordinates (including, without limitation, those sets depositedat the Research Collaboratory for Structural Bioinformatics Protein DataBank (PDB) with the accession numbers PDB ID: 1FFK; PDB ID: 1FF2; PDBID: 1FG0; and PDB ID: 1JJ2, and/or contained on Disk No. 1, 2, or 3)referred to herein is incorporated by reference herein.

All materials submitted herewith on Disk Nos. 1, 2 and 3 areincorporated by reference herein. Disk Nos. 1, 2 and 3 are identified ascontaining the following files:

Disk No. 1 of 3: File Name: Size: Date Created 1) 1ffk.doc 7,046 KB Jul.25, 2001 2) 1ffk.ent 5,484 KB Jul. 25, 2001 3) 1ffz.doc 1,219 KB Jul.25, 2001 4) 1ffz.ent 937 KB Jul. 25, 2001 5) 1fg0.doc 1,225 KB Jul. 25,2001 6) 1fg0.ent 942 KB Jul. 25, 2001 7) <dir> Jul. 25, 2001 8) <dir>Jul. 25, 2001

Disk No. 2 of 2: File Name: Size: Date Created: 1) 1jj2.rtf 12,742 KBJul. 25, 2001 2) 1jj2.txt 8,372 KB Jul. 26, 2001 3) <dir> Jul. 26, 20014) <dir> Jul. 26, 2001

Disk No. 3 of 3: File Name: Size: Date Created: 1) anisomycin.pdb 7,593KB Jul. 18, 2001 2) blasticidin.pdb 7,594 KB Jul. 18, 2001 3)carbomycin.pdb 7,592 KB Jul. 18, 2001 4) sparsomycin.pdb 7,541 KB Jul.18, 2001 5) spiramycin.pdb 7,592 KB Jul. 18, 2001 6) tylosin.pdb 7,592KB Jul. 18, 2001 7) virginiamycin.pdb 7,591 KB Jul. 18, 2001 8) <dir>Jul. 18, 2001 9) <dir> Jul. 18, 2001Equivalents

The invention may be embodied in other specific forms without departingform the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

1-46. (canceled)
 47. A computer system comprising: (a) a memory havingstored therein data indicative of atomic co-ordinates derived from anelectron density map having a resolution of at least about 4.5 Å anddefining a ribofunctional locus of a large subunit of a ribosome; and(b) a processor in electrical communication with the memory, theprocessor comprising a program for generating a three-dimensional modelrepresentative of the ribofunctional locus. 48-75. (canceled)
 76. Amethod of identifying a candidate molecule, the method comprising thesteps of: (a) providing a molecular model of a ribofunctional locus of alarge subunit of a ribosome, wherein the molecular model is based onatoms derived from an electron density map having a resolution of atleast about 4.5 Å; and (b) using the model to identify a candidatemolecule having a surface complementary to the ribofunctional locus. 77.The method of claim 76, wherein the candidate molecule binds theribofunctional locus of the large subunit of the ribosome.
 78. Themethod of claim 76, comprising the additional step of producing thecandidate molecule identified in step (b).
 79. The method of claim 76,comprising the additional step of determining whether the candidatemolecule modulates ribosomal activity.
 80. The method of claim 79,comprising the additional step of identifying a modified molecule. 81.The method of claim 80, comprising the additional step of producing themodified molecule.
 82. The method of claim 81, comprising the additionalstep of determining whether the modified molecule modulates ribosomalactivity.
 83. The method of claim 82, comprising the additional step ofproducing the modified molecule.
 84. The method of claim 76, wherein thecandidate molecule is an antibiotic or an antibiotic analogue.
 85. Themethod of claim 80, wherein the modified molecule is an antibiotic or anantibiotic analogue.
 86. The method of claim 84, wherein the antibioticor antibiotic analogue is a macrolide.
 87. The method of claim 76,wherein the ribofunctional locus comprises at least a portion of anactive site.
 88. The method of claim 87, wherein the active sitecomprises at least a portion of a peptidyl transferase site.
 89. Themethod of claim 87, wherein the peptidyl transferase site is defined bya plurality of residues set forth in Table
 5. 90. The method of claim76, wherein the ribofunctional locus comprises at least a portion of anA-site.
 91. The method of claim 90, wherein the A-site is defined by aplurality of residues set forth in Table
 6. 92. The method of claim 76or 90, wherein the ribofunctional locus comprises a least a portion of aP-site.
 93. The method of claim 92, wherein the P-site is defined by aplurality of residues set forth in Table
 7. 94. The method of claim 76,wherein the ribofunctional locus comprises at least a portion of apolypeptide exit tunnel.
 95. The method of claim 94, wherein the exittunnel is defined by a plurality of residues set forth in Table 8, Table9 or Table
 10. 96. The method of claim 92, wherein the ribofunctionallocus comprises at least a portion of a polypeptide exit tunnel.
 97. Themethod of claim 96, wherein the exit tunnel is defined by a plurality ofresidues set forth in Table 8, Table 9 or Table
 10. 98. The method ofclaim 76, wherein the ribofunctional locus is defined by a plurality ofresidues set forth in Table 11, Table 12, Table 13, Table 14, Table 15,Table 16 or Table
 17. 99. The method of claim 76, wherein the molecularmodel is in an electronic form.
 100. The method of claim 76, wherein themolecular model is generated from atomic co-ordinates produced bymolecular modeling.
 101. The method of claim 76, wherein the molecularmodel is generated from atomic co-ordinates produced by homologymodeling using at least a portion of the atomic co-ordinates depositedat the Protein Data Bank under accession number PDB ID: 1FFK, 1FFZ,1FG0, or 1JJ2.
 102. The method of claim 76, wherein the molecular modelis generated from atomic co-ordinates produced by molecular replacementusing at least a portion of the atomic co-ordinates deposited at theProtein Data Bank under accession number PDB ID: 1FFK, 1FFZ, 1FG0, or1JJ2.
 103. The method of claim 76, wherein the molecular model comprisesresidues that are conserved among prokaryotic organisms.
 104. The methodof claim 76, wherein the molecular model comprises a residue that ispresent in a prokaryotic ribosome but is absent from a eukaryoticribosome.
 105. The method of claim 104, wherein the eukaryotic ribosomeis a mammalian ribosome. 106-112. (canceled)